A method is described for the identification and cloning of promoters that express under a defined environmental condition, such as growth in glucose medium. Using this method, five Trichodermal promoters capable of the high expression of operably linked coding sequences are identified, one of which is the promoter for T. reesei tef1. Also provided are altered cbh1 promoters, altered so that glucose no longer represses expression from such promoter. The invention further provides vectors and hosts that utilize such promoters, and unique fungal enzyme compositions from such hosts.
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22. A promoter, wherein said promoter is selected from the cbh1 promoter of the group consisting of SEQ ID 19, SEQ ID 20, SEQ ID 21 and SEQ ID 22.
35. A glucose-derepressed promoter selected from the group consisting of the altered cbh1 promoter contained in DSM 11964, the altered cbh1 promoter contained in DSM 11965, and the altered cbh1 promoter contained in DSM 11966.
1. An isolated promoter capable of expressing an operably-linked coding sequence in a fungal host grown on glucose wherein said promoter is selected from the group consisting of the Trichoderma tef1 promoter, the promoter of SEQ ID No. 2, the promoter of SEQ ID No. 3, the promoter of SEQ ID No. 4, the promoter of SEQ ID No. 5, the promoter of SEQ ID No. 6 and a derepressed cbh1 promoter, wherein said derepressed cbh1 promoter contains sequence alterations, relative to the native T. reesei cbh1 promoter (SEQ ID NO: 18), and wherein said derepressed cbh1 promoter has an increased ability to express said coding sequence when said fungal host is grown on said glucose relative to the ability of the native T. reesei cbh1 promoter to express said coding sequence when said same fungal host is grown on said glucose.
2. The promoter of
a. growing said host on glucose; b. extracting mRNA from said host; c. preparing a cDNA bank from a first sample of said mRNA; d. detectably labelling a sample of said cDNA; e. hybridizing said labelled cDNA to said cDNA bank; f. selecting clones from said hybridization of step (e) on the basis of the intensity of the hybridization; g. determining the relative abundancy of said selected clones in the cDNA bank of step (c); h. identifying the most abundant clones of step (g); and i. using the inserts of the clones of step (h) to identify and clone the host promoter.
4. The promoter of
6. The promoter of
9. The promoter of
16. The promoter of
20. The promoter of
21. The promoter of
23. A vector comprising the promoter of--any one of
25. The vector of
27. The vector of
31. The host cell of
33. The host cell of
36. The glucose-derepressed promoter of
37. The glucose-derepressed promoter of
38. The glucose-derepressed promoter of
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This application is a division of U.S. application Ser. No. 08/389,564, filed Feb. 16, 1995, which is a continuation of U.S. application Ser. No. 07/932,485, filed Aug. 19, 1992 (abandoned), which is a continuation-in-part of U.S. application Ser. No. 07/496,155, filed Mar. 19, 1990, which is a continuation of U.S. application Ser. No. 07/044,077, filed Apr. 29, 1987 (abandoned).
I. Methods for the Identification of Promoters
Many systems have been used to isolate genes and their promoters located immediately upstream of the translation start site of a gene. The techniques can roughly be divided in two categories, namely (1) where the aim is to isolate genomic DNA fragments containing promoter activity randomly by so-called promoter probe vector systems and (2) where the aim is to isolate a gene per se from a genomic bank (library) and isolation of the corresponding promoter follows therefrom.
In promoter probe vector systems, genomic DNA fragments are randomly cloned in front of the coding sequence of a reporter gene that is expressed only when the cloned fragment contains promoter activity (Neve, R. L. et al., Nature 277:324-325 (1979)). Promoter probe vectors have been designed for cloning of promoters in E. coli (An, G. et al., J. Bact. 140:400-407 (1979)) and other bacterial hosts (Band, L. et al., Gene 26:313-315 (1983); Achen, M. G., Gene 45:45-49 (1986)), yeast (Goodey, A. R. et al., Mol. Gen. Genet. 204:505-511 (1986)) and mammalian cells (Pater, M. M. et al., J. Mol. App. Gen. 2:363-371 (1984)). Because it is well known in the art that Trichoderma promoters fail to work in E. coli and yeast (e.g. Penttila, M. E. et al., Mol. Gen. Genet. 194:494-499 (1984)), these organisms cannot be used as hosts to isolate Trichoderma promoters. Due to the fact that, during the transformation of Trichoderma, the transforming DNA integrates into the fungal genome in varying copies in random locations, application of this method by using Trichoderma itself as a cloning host is also unlikely to succeed and would not be practical for efficient isolation of Trichoderma promoters with the desired properties.
Known genes can be isolated from either a cDNA or chromosomal gene bank (library) using hybridization as a detection method. Such hybridization may be with a corresponding, homologous gene from another organism (e.g. Vanhanen et al., Curr. Genet. 15:181-186 (1989)) or with a probe designed on the basis of expected similarities in amino acid sequence. If amino acid sequence is available for the corresponding protein, an oligonucleotide can also be designed which can be used in hybridization for isolation of the gene. If the gene is cloned into an expression bank, the expression product of gene can be also detected from such expression bank by using specific antibodies or an activity test.
Specific genes can be isolated by using complementation of mutations in E. coli or yeast (e.g. Keesey, J. K. et al., J. Bact. 152:954-958 (1982); Kaslow, D. C., J. Biol. Chem. 265:12337-12341 (1990); Kronstad, J. W., Gene 79:97-106 (1989)), or complementation of corresponding mutants of filamentous fungi for instance by using SIB selection (Akins et al., Mol. Cell. Biol. 5:2272-2278 (1985)).
However, a major concern is how to isolate specific genes that have the desired promoter properties, for example genes which would be most highly expressed when glucose is present in the medium. There is no information available in the literature to indicate which genes are the most highly expressed in an organism, and especially not from filamentous fungi. The phosphoglyceratekinase (PGK) promoter from the yeast Saccharomyces cerevisiae is considered to be a strong promoter for protein production. However, results obtained by the inventors have shown that the corresponding Trichoderma promoter is not suitable for such protein production. Thus, the identification of specific Trichoderma genes for their isolation in order the best possible promoter for protein production in certain desired conditions is unknown and cannot be predicted. Consequently one cannot rely on any previous nucleotide or amino acid sequence information, nor complement any previously known mutations, in gene isolation for such purpose in Trichoderma.
Differential hybridization has been used for cloning of genes expressed under certain conditions. The method relies on the screening of a bank separately with an induced and noninduced cDNA probe. By this method e.g. Trichoderma reesei genes strongly expressed during production of cellulolytic enzymes have been isolated (Teeri, T. et al., Bio/Technology 1:696-699 (1983)). The differential hybridization methods used are based on the idea that the genes searched for are expressed in certain conditions (like cellulases on cellulose) but not in some other conditions (like cellulases on glucose) which enables picking up clones hybridizing with only one of the cDNA probes used. However, for isolation of the genes expressed strongly on glucose, this approach (expression on glucose and not on some other media) is not a suitable one, and might in fact result in not finding the most highly expressed genes. This is because when differentially screening a chromosomal bank, only induced genes are selected. Such induced genes are not necessarily the most strongly expressed genes. Thus, no method is known in the art which would permit the identification of promoters which function strongly in Trichoderma on glucose medium.
Another option for obtaining a promoter with desired properties is to modify the already existing ones. This is based on the fact that the function of a promoter is dependent on the interplay of regulatory proteins which bind to specific, discrete nucleotide sequences in the promoter, termed motifs. Such interplay subsequently affects the general transcription machinery and regulates transcription efficiency. These proteins are positive regulators or negative regulators (repressors), and one protein can have a dual role depending on the context (Johnson, P. F. and McKnight, S. L. Annu. Rev. Biochem. 58:799-839 (1989)). However, even a basic understanding of the regions responsible for regulation of a promoter requires a considerable amount of experimental data, and data obtained from the corresponding promoter of another organism is usually not useful (see Vanhanen, S. et al., Gene 106:129-133 (1991)), or at least not sufficient, to explain the function of a promoter originating from another organism.
II. Translation Elongation Factors
Translation Elongation Factors (TEFs) are universally conserved proteins that promote the GTP-dependent binding of an aminoacyl-tRNA to ribosomal A-site in protein synthesis. Especially conserved is the N-terminus of the protein containing the GTP binding domain. TEFs are known as very abundant proteins in cells comprising about 4-6% of total soluble proteins (Miyajima, I. et al., J. Biochem. 83:453-462 (1978); Thiele, D. et al., J. Biol. Chem. 260:3084-3089 (1985)).
tef genes have been isolated from several organisms. In some of them they constitute a multigene family. Also a number of pseudogenes have been isolated from some organisms. The promoter of the human tef gene can direct transcription in vitro at least 2-fold more effectively than the adenovirus major late promoter, which indicates that the tef promoter is a strong promoter in mammalian expression systems (Uetsuki et al., J. Biol. Chem. 264:5791-5798 (1989)). Both the human and the A. thaliana tef1 promoter (for translation elongation factor EF-1α) has been used in an expression system with high efficiency of gene expression (Kim et al., Gene 91:217-223 (1990); Curie et al., Nucl. Acid Res. 19:1305-1310 (1991)). In both cases the full expression of the promoter was dependent on the presence of the intron in the 5' noncoding region.
tef is quite constitutively expressed, the major exception being its expression in aging and quiescent cells. It is not known to be regulated by the growth substrates of the host.
III. Expression of Recombinant Proteins in Trichoderma
The filamentous fungus Trichoderma reesei is an efficient producer of hydrolases, especially of different cellulose degrading enzymes. Due to its excellent capacity for protein secretion and developed methods for industrial cultivations, Trichoderma is a powerful host for production of heterologous, recombinant proteins in large scale. The efficient production of both homologous and heterologous proteins in fungi relies on fungal promoters. The promoter of the main cellulase gene of Trichoderma, cellobiohydrolase 1 (cbh1), has been used for production of heterologous proteins in Trichoderma grown on media containing cellulose or its derivatives (Harkki et al., Bio/Technology 7:596-603 (1989); Saloheimo et al., Bio/Technology 9:987-990 (1991)). The cbh1 promoter cannot be used when the Trichoderma are grown on glucose containing media due to glucose repression of cbh1 promoter activity. This regulation occurs at the transcriptional level and thus glucose repression could be mediated through the promoter sequences. However, nothing is yet known of the mechanism of glucose repression at the promoter level in filamentous fungi.
Glucose repression in the yeast Saccharomyces cerevisiae has been studied for many years. These studies have however failed, until recently, to identify binding sequences in promoters or regulatory proteins binding to promoters which would mediate glucose repression. The first ever published glucose repressor protein and the binding sequence in eukaryotic cells was published by Nehlin and Ronne (Nehlin, J. O. and Ronne, H. EMBO J. 9:2891-2899 (1990)). This MIG1 protein seems to be responsible of one fifth of the glucose repression of GAL genes in Saccharomyces cerevisiae, other factors still being required to obtain full glucose repression effect (Nehlin, J. O. et al., EMBO J. 10:3373-3377 (1991)).
Thus, it is desirable to be able to produce proteins in Trichoderma grown on glucose. Not only is the substrate glucose cheap and readily available, but also Trichoderma produces less protease activity when grown on glucose. Further, cellulase production is repressed when Trichoderma is grown on glucose, thus allowing for the easier purification of the desired product from the Trichoderma medium. Nevertheless, to date there has been no identification or characterization of any promoter that is highly functional in Trichoderma grown on glucose. In addition, no modifications of the normally glucose repressed promoter, the cbh1 promoter, have been identified which would allow the use of this strong promoter for expression of heterologous genes in Trichoderma grown on glucose.
This invention is first directed to the identification of the motif, the DNA element, that imparts glucose repression onto the Trichoderma cbh1 promoter.
The invention is further directed to a modified Trichoderma cbh1 promoter, such modified promoter lacking such glucose repression element and such modified promoter being useful for the production of proteins, including cellulases, when the host is grown on glucose medium.
The invention is further directed to a method for the isolation of genes that are highly expressed on glucose, especially from filamentous fungal hosts such as Trichoderma.
The invention is further directed to five such previously undescribed genes and their promoters from Trichoderma reesei;
The invention is further directed to specific cloning vectors for Trichoderma containing the above mentioned sequences.
The invention is further directed to filamentous fungal strains transformed with said vectors, which strains thus are able to produce proteins such as cellulases on glucose.
The invention is further directed to a process for producing cellulases or other useful enzymes on glucose.
FIG. 1. FIG. 1A shows the plasmid pTHN1 which carries the tef1 promoter and 5' part of the coding region and shows the relevant features of the tef1 gene and the sequenced areas. FIGS. 1B-1D show is the nucleotide sequence of the tef1 promoter and coding sequence [SEQ ID 1]. The promoter sequence stops at base number 1234. The methionine codon of the start site of translation is located at base numbers 1235-1237 and is underlined. The total number of bases is 3461. The DNA sequence composition is 850 A; 1044 C; 860 G; 697 T; and 10 other. The sequence name is TEF001.
FIG. 2 shows the plasmid pEA33 which carries the tef1 promoter and the coding region with relevant features.
FIG. 3. FIG. 3A shows the plasmid pTHN3 which carries the promoter and coding region of the clone cDNA1 and shows the relevant features. FIG. 3B is the nucleotide sequence of the cDNA1 promoter and coding sequence [SEQ ID 2]. The promoter sequence stops at base number 1857. The methionine codon of the start site of translation is located at base numbers 1858-1860 and is underlined. The total number of bases is: 2336. The DNA sequence composition is 582 A; 618 C; 596 G; 535 T; and 5 other. The first 700 bases shown in SEQ ID No:2 are vector pSP73 sequences.
FIG. 4. FIG. 4A shows the plasmid pEA10 which carries the promoter and coding region of the clone cDNA10 and the relevant regions and sequenced areas. Diagonally hatched=insert; solid line=sequenced region (genomic DNA); squared criss-crossed=sequenced region (cDNA). Not all EcoRV and NdeI sites are shown. FIGS. 4B and 4C show the nucleotide sequence of the cDNA10 promoter and coding sequence [SEQ ID 3]. The promoter sequence stops at base number 1522. The methionine codon of the start site of translation is located at base numbers 1523-1525 and is underlined. The total number of bases is: 2868. The DNA sequence composition is: 760 A; 765 C; 675 G; 668 T; and 0 other. The sequence name is CDNA10SEQ.
FIG. 5. FIG. 5A shows the plasmid pEA12 which carries the clone cDNA12 and relevant features and sequenced areas. Diagonally hatched=insert; solid line=sequenced region (genomic DNA); squared criss-crossed=sequenced region (cDNA). ?=unsequenced intron region. Note: AvaI is not a unique site. FIGS. 5B and 5C show the nucleotide sequence of the cDNA12 promoter and coding sequence [SEQ ID 4]. The promoter sequence stops at base number 1101. The methionine codon of the start site of translation is located at base numbers 1102-1104 and is underlined. The total number of bases is: 2175. The DNA sequence composition is: 569 A; 602 C; 480 G; 519 T; and 5 other. The sequence name is A12DNA.
FIG. 6. FIG. 6A shows the plasmid pEA155 which carries the promoter and coding region of the clone cDNA15 and the relevant features and sequenced areas. Diagonally hatched=insert; solid line=sequenced region (genomic DNA); squared criss-crossed=sequenced region (cDNA). Not all PstI and EcoRI sites are shown. FIGS. 6B and 6C show the nucleotide sequence of the cDNA15 promoter and coding sequence [SEQ ID 5]. The total number of bases is: 2737. The DNA sequence composition is: 647 A; 695 C; 742 G; 649 T; and 4 other. The sequence name is A15DNA
FIG. 7. FIG. 7A shows plasmid pPLE3 which carries the egl1 cDNA. Just above the plasm id map is the sequence of the adaptor molecule [SEQ ID NO: 25] that was constructed to remove the small SacII and Asp718 fragment from the plasmid so as to construct an exact joint [SEQ ID NO: 26, SEQ ID NO: 27] between the cbh1 promoter and the egl1 signal sequences [SEQ ID NO:18 and 16]. FIG. 7B shows the sequence of the egl1 cDNA [SEQ ID NO:16]. The total number of bases is: 1588. The DNA sequence composition is: 369 A; 527 C; 418 G; and 274 T. FIG. 7C shows the sequence of the cbh1 terminator of pPLE3 [SEQ ID NO:23]. The total number of bases is: 745. The DNA sequence composition is: 198 A; 191 C; 177 G; and 0 T.
FIG. 8 shows construction of plasmid pEM-3A. The "A" on the plasmid maps denotes the EGI tail sequence and the "B" denotes the EGI hinge sequence. SEQ ID NO:28 is also shown in the figure.
FIG. 9 shows the plasmid pTHN100B for expression of the EGIcore under the tef1 promoter. SEQ ID NO:28 is also shown in the figure.
FIG. 10 shows production of EGIcore from the plasmid pTHN100B into the culture medium of the host strain QM9414 analyzed by EGI specific antibodies from a slot blot. Lane 1: pTHN100B-16b, 200 μl glucose supernatant; lane 2: QM9414, 200 μl glucose supernatant; lane 3: TBS; lane 4: QM9414, 200 μl solka floc 1:500 diluted supernatant; lane 5: QM9414, 200 μl solka floc 1:5,000 diluted supernatant; lane 6: QM9414, 200 μl solka floc 1:10,000 diluted supernatant; lane 7: pTHN100B-16b, 200 μl glucose 1:5 diluted supernatant; lane 8: QM9414, 200 μl glucose 1:5 diluted supernatant; lane 9: 200 ng EGI protein; lane 10: 100 ng EGI protein; lane 11: 50 ng EGI protein; and lane 12: 25 ng EGI protein.
FIG. 11 shows Western blotting with EGI specific antibodies of culture medium of the strain pTHN100B-16c grown in whey-spent grain or glucose medium, and of EGIcore purified from the glucose medium. Lane 1: pTNH100B-16c, 10 μl whey spent grain supernatant; lane 2: pTNH100B-16c, 5 μl whey spent grain supernatant; lanes 3-5: EGIcore purified from pTHN100B-16c glucose fermentation; lane 6: pTHN100B-16c, 15 μl glucose fermenter supernatant, concentrated 100×; lane 7: pTHN100B-16c, 7.5 μl glucose fermenter supernatant, concentrated 100×; and lane 8: low molecular weight markers at 94 kDa, 67 kDa, 43 kDa, 30 kDa and 20.1 kDa (bands 1-5 starting from lane 8, top of gel).
FIG. 12 shows Western blotting of culture medium of the strain pTHN100B-16c grown on glucose medium. Lane 1: EGI protein, about 540 ng; lane 2, EGI protein, about 220 ng; lane 3, EGI protein, about 110 ng; lane 4: pTHN100B-16c, 30 μl glucose fermenter supernatant; lane 5: pTHN100B-16c, 30 μl glucose fermenter supernatant, concentrated 4.2×; lane 6: low molecular weight markers at 94 kDa, 67 kDa, 43 kDa, 30 kDa and 20.1 kDa (bands 1-5 starting from lane 6, top of gel).
FIG. 13. FIG. 13A diagrams the elements of the plasmid pMLO16. FIGS. 13B and 13C show the sequence of the cbh1 promoter of plasmid pML016 [SEQ ID18]. FIGS. 13D and 13E show the sequence of the T. reesei cbh1 terminator on plasmid pML016 and plasmids derived from it [SEQ ID24]. The total number of bases is 2218. The DNA sequence composition is: 600 A; 501 C; 550 G; 567 T; and 0 other.
FIG. 14 shows the expression of β-galactosidase on glucose medium in pMLO16de15(11)-transformants of Trichoderma reesei QM 9414 (A2-F5). A1: QM 9414 host strain; C1 and E1: QM 9414 transformant in which one copy of β-galactosidase expression cassette with intact cbh1 promoter has replaced the cbh1 locus; B1, D1 and F1: empty wells.
FIG. 15. FIG. 15A shows the restriction map of the plasmid pMLO16de15(11), which carries the shortened form of the cbh1 promoter fused to the lacZ gene and the cbh1 terminator. FIG. 15B is the sequence of the truncated cbh1 promoter [SEQ ID19]. The polylinker is underlined. The arrow denotes the deletion site.
FIG. 16. FIG. 16A shows the restriction map of the plasmid pMLO17, which carries the shortened form of the cbh1 promoter fused to the cbh1 chromosomal gene. The restriction sites marked with a superscripted cross "+ " are not single sites. There are two additional EcoRI sites in the cbh1 gene that are not shown. FIGS. 16B and 16C show the sequence of the KspI-XmaI fragment (the underlined portion) that contains the chromosomal cbh1 gene [SEQ ID17].
FIG. 17A shows the expression of CBHI on glucose medium in pMLO17 transformants of Trichoderma reesei QM 9414. A collection of single spore cultures (number and a letter-code) (FIG. 17B) and different control samples are shown.
FIG. 18. FIG. 18A shows specific mutations of mig-like sequences (M) in cbh1 promoters of pMI-24, pMI-25, pMI-26, pMI-27 and pMI-28. The promoters shown here were fused to lacZ gene and cbh1 terminator as described for pMLO16 (see FIG. 13) or pMLO16de10(2) (see FIG. 19). *: sequence alteration made in cbh1 promoter in different combinations. At position -1510 to -1505 the genomic sequence is 5'-CTGGGG and the altered sequence is 5'-TCTAAA. At position -1006 to -1001 the genomic sequence is 5'-CTGGGG and the altered sequence is 5'-TCTAAA. At position -725 to -720 the genomic sequence is 5'-GTGGGG and the altered sequence is 5'-TCTAGA. At position -699 to -694 the genomic sequence is 5'-CCCCAC and the altered sequence is 5'-CCCAC. At position -691 to -686 the genomic sequence is 5'-CCCCAC and the altered sequence is 5'-ACCCAC. pMLO16de10(2) was used as a starting vector for pMI-25, pMI-26, pMI-27 and pMI-28, pMLO16 for pMI-24. v=the polylinker. FIGS. 18B and 18C show the sequence of the altered cbh1 promoter of pMI-24 ([SEQ ID20]). The polylinker is underlined and the sequence alteration is boxed. FIGS. 18D and 18 E show the sequence of the altered cbh1 promoter of pMI-27 ([SEQ ID21]). The polylinker is underlined, the arrow denotes the deletion point and the sequence alterations are boxed. The total number of bases is 1781. The DNA sequence composition is: 487 A; 402 C; 435 G; 457 T; and 0 other. Comments: M127 PROM SEQ ID21 GLC-PROM-PAT MI. 15.8.1992. The sequence name is PMI27PROM. FIGS. 18F and 18G show the sequence of the altered cbh1 promoter of pMI-28 ([SEQ ID221]). The total number of bases is 1781. The DNA sequence composition is: 490 A; 402 C; 431 G; 458 T; and 0 other. Comments: MI28 PROM SEQ ID22 GLC-PROM-PAT MI. 15.8.1992. The sequence name is PMI28PROM. The polylinker is underlined, the arrow denotes the deletion point and the sequence alterations are boxed.
FIG. 19 shows the restriction map of the plasmid pMLO16de10(2), which carries the shortened form of the cbh1 promoter fused to lacZ gene and the cbh1 terminator.
FIG. 20 shows the expression of β-galactosidase on indicated medium in Trichoderma reesei QM9414 transformed with pMLO16de10(2), pMI-25, pMI-27, pMI-28, pMLO16 and pMI-24.
I. Identification of Fungal Genes that Express on Glucose Medium
In the following description, reference will be made to various methodologies known to those of skill in the art of molecular genetics and biology. Publications and other materials setting forth such known methodologies to which reference is made are incorporated herein by reference in their entireties as though set forth in full.
General principles of the biochemistry and molecular biology of the filamentous fungi are set forth, for example, in Finkelstein, D. B. et al., eds., Biotechnology of Filamentous Fungi: Technology and Products, Butterworth-Heinemann, publishers, Stoneham, Mass. (1992) and Bennett, J. W. et al., More Gene Manipulations in Fungi, Academic Press--Harcourt Brace Jovanovich, publishers, San Diego Calif. (1991).
To be able to develop versatile systems for protein production from Trichoderma, especially when Trichoderma are grown on glucose, a method has been developed for the isolation of previously unknown Trichoderma genes which are highly expressed on glucose, and their promoters. The method of the invention requires the use of only one cDNA population of probes.
It is to be understood that the method of the invention would be useful for the identification of promoter sequences that are active under any desired environmental condition to which a cell could be exposed, and not just to the exemplified isolation of promoters that are capable of expression in glucose medium. By "environmental condition" is meant the presence of a physical or chemical agent, such agent being present in the cellular environment, either extracellularly or intracellularly. Physical agent would include, for example, certain growth temperatures, especially a high or low temperature. Chemical agents would include any compound or mixtures including carbon growth substrates, drugs, atmospheric gases, etc.
According to the method of the invention, the organism is first grown under the desired growth condition, such as the use of glucose as a carbon source. Total mRNA is then extracted from the organism and preferably purified through at least a polyA+ enrichment of the mRNA from the total RNA population. A cDNA bank is made from this total mRNA population using reverse transcriptase and the cDNA population cloned into any appropriate vector, such as the commercially available lambda-ZAP vector system (Stratagene). When using the lambda-ZAP vector system, or any lambda vector system, the cDNA is packaged such that it is suitable for infection of any E. coli strain susceptable to lambda bacteriophage infection.
The cDNA bank is transferred by standard colony hybridization techniques onto nitrocellulose filters for screening. The bank is plated and plaque lifts are taken onto nitrocellulose. The bank is screened with a population of labelled cDNAs that had been synthesized against the same RNA population from which the cloned cDNA bank was constructed, using stringent hybridization conditions. It should be noted that the genes are not expressed in any way during this selection process. This results in clones hybridizing with varying intensity and the ones showing the strongest signals are picked. Genes that are most strongly expressed in the original population comprise the majority of the total mRNA pool and thus give a strong signal in this selection.
The inserts in clones with the strongest signals are sequenced from the 3' end of the insert using any standard DNA sequencing technique as known in the art. This provides a first identification of each clone and allows the exclusion of identical clones. The frequency with which each desired clone is represented in the cDNA lambda-bank is determined by hybridizing the bank against a clone-specific PCR probe. The desired clones are those which, in addition to having the strongest signals as above, are also represented at the highest frequencies in the cDNA bank, since this implies that the abundancy of the mRNA in the population was relatively high and thus that the promoter for that gene was highly active under the growth conditions. Thus, the relevance of this approach and any clone identified therefrom can be double-checked: the intensity of the hybridization signal of a specific clone should correlate positively with the frequency with which that clone is found in the cDNA bank. The inserts of the clones selected in this manner, such inserts corresponding to the cDNA sequences, may be used as probes to isolate the corresponding genes and their promoters from a chromosomal bank, such as one cloned into lambda as above.
The method of the invention is not limited to Trichoderma, but would be useful for cloning genes from any host, or from a specific tissue with such host, from which a cDNA bank may be constructed, including, prokaryote (bacterial) hosts, and any eukaryotic host plants, mammals, insects, yeast, and any cultured cell populations.
For example, using the method of the invention, five genes that express relatively high levels of mRNA in Trichoderma reesei when such Trichoderma are grown on glucose were identified. These genes were sequenced and identified as clone cDNA33, cDNA1, cDNA10, cDNA12, and cDNA15. When used to screen a Trichoderma chromosomal lambda-bank, the corresponding genes and their promoters were identified. Such genes and promoters (or portions thereof) may then be subcloned into any desired vector, such as the pSP73 vector (Promega, Madison, Wis., USA).
According to the invention, the clones containing the genes and their promoters (or parts of them) highly expressed in Trichoderma grown on glucose are represented as follows:
| ______________________________________ |
| Plasmid FIG. cDNA FIG. SEQ ID NO: |
| ______________________________________ |
| pTHN1 1A cDNA33 1B-1D 1 |
| pEA33 2 cDNA33 1B-1D 1 |
| pTHN3 3A cDNA1 3B 2 |
| pEA10 4A cDNA10 4B, 4C 3 |
| pEA12 5A cDNA12 5B, 5C 4 |
| pEA155 6A cDNA15 6B, 6C 5 |
| ______________________________________ |
One of the genes isolated according to the invention as being highly expressed when Trichoderma was grown on glucose has been identified as the one encoding Trichoderma translation elongation factor 1α (tef 1). In addition, four other, new genes have been identified for the first time that are highly expressed on glucose in Trichoderma.
These data show that the method used in this invention resulted in isolating five genes, one of which (tef1) is known to be efficiently expressed in other organisms. However, the tef1 gene was not the most highly expressed of the five genes isolated from the Trichoderma cDNA bank by the method of the invention.
Of the five genes isolated, only tef1 shows a relevant degree of homology to any known protein sequences. All of the genes isolated are also expressed on other carbon sources and would not have been found with the classical method of differential cloning. This shows the importance of the method used in this invention in isolation of the most suitable genes for a specific purpose, such as for isolation of strong promoters for expression on glucose containing medium.
The promoter of any of these genes may be operably linked to a sequence heterologous to such promoter, and especially heterologous to the host Trichoderma, for expression of such gene from a Trichoderma host that is grown on glucose. Preferably, the coding sequence provides a secretion signal for secretion of the recombinant protein into the medium.
Use of the promoters of the invention allow for the expression of genes from Trichoderma under conditions in which there are no cellulases and relatively few proteases. Thus, for the first time, recombinant genes can be highly expressed on Trichoderma using a glucose-based growth medium.
The promoters of the invention, while being strongly expressed on glucose (that is, when the filamentous fungal host is grown on medium providing glucose as a carbon and energy source), are not repressed in the absence of glucose. In addition, they are active when the Trichoderma host is grown on carbon sources other than glucose.
The glucose promoters of the invention, and those identified by the methods of the invention, can be used to produce enzymes native to Trichoderma itself, especially of those capable of hydrolysing different kinds of plant material. On glucose, the fungus does not naturally produce these enzymes and consequently one or more specific hydrolytic enzymes could be produced on glucose medium free from other plant material hydrolyzing enzymes. This would result in an enzyme preparation or enzyme mixtures for specific applications.
II. Modification of the Cellobiohydrolase I Promoter
This invention also describes a method for the modification of the cellobiohydrolase 1 promoter (cbh1) such that the activity of the promoter is retained but the promoter no longer is repressed when cells are grown on glucose-containing medium. Essentially, the DNA motif that imparted glucose repression has been identified and removed from this promoter, allowing production of desired proteins whose coding sequences are operably linked to the promoter in suitable hosts, such as Trichoderma. Such a modified cbh1 promoter is termed a derepressed cbh1 promoter. As above, when the recombinant organisms obtained from transformation with such constructs are cultivated on glucose containing medium, any protein, including a cellulase may be produced without production of other plant material hydrolysing enzymes, especially of native cellulases.
Isolated glucose promoters or derepressed cbh1 promoter can be used for instance to produce separate individual cellulases in hosts grown on glucose without any simultaneous production of other hydrolases such as other cellulases, hemicellulases, xylanases etc. or to produce heterologous proteins in varying growth media.
III. Preparation of Coding Sequences Operably Linked to the Promoter Sequences of the Invention
The process for genetically engineering a coding sequence, for expression under a promoter of the invention, is facilitated through the isolation and partial sequencing of pure protein encoding an enzyme of interest or by the cloning of genetic sequences which are capable of encoding such protein with polymerase chain reaction technologies; and through the expression of such genetic sequences. As used herein, the term "genetic sequences" is intended to refer to a nucleic acid molecule (preferably DNA). Genetic sequences that are capable of encoding a protein are derived from a variety of sources. These sources include genomic DNA, cDNA, synthetic DNA, and combinations thereof. The preferred source of genomic DNA is a fungal genomic bank. The preferred source of the cDNA is a cDNA bank prepared from fungal mRNA grown in conditions known to induce expression of the desired gene to produce mRNA or protein. However, since the genetic code is universal, a coding sequence from any host, including prokaryotic (bacterial) hosts, and any eukaryotic host plants, mammals, insects, yeasts, and any cultured cell populations would be expected to function (encode the desired protein).
Genomic DNA may or may not include naturally occurring introns. Moreover, such genomic DNA may be obtained in association with the 5' promoter region of the gene sequences and/or with the 3' transcriptional termination region. According to the invention however, the native promoter region would be replaced with a promoter of the invention.
Such genomic DNA may also be obtained in association with the genetic sequences which encode the 5' non-translated region of the mRNA and/or with the genetic sequences which encode the 3' non-translated region. To the extent that a host cell can recognize the transcriptional and/or translational regulatory signals associated with the expression of the mRNA and protein, then the 5' and/or 3' non-transcribed regions of the native gene, and/or, the 5' and/or 3' non-translated regions of the mRNA may be retained and employed for transcriptional and translational regulation.
Genomic DNA can be extracted and purified from any host cell, especially a fungal host cell, which naturally expresses the desired protein by means well known in the art. A genomic DNA sequence may be shortened by means known in the art to isolate a desired gene from a chromosomal region that otherwise would contain more information than necessary for the utilization of this gene in the hosts of the invention. For example, restriction digestion may be utilized to cleave the full-length sequence at a desired location. Alternatively, or in addition, nucleases that cleave from the 3'-end of a DNA molecule may be used to digest a certain sequence to a shortened form, the desired length then being identified and purified by gel electrophoresis and DNA sequencing. Such nucleases include, for example, Exonuclease III and Bal31. Other nucleases are well known in the art.
For cloning into a vector, such suitable DNA preparations (either genomic DNA or cDNA) are randomly sheared or enzymatically cleaved, respectively, and ligated into appropriate vectors to form a recombinant gene (either genomic or cDNA) bank.
A DNA sequence encoding a desired protein or its functional derivatives may be inserted into a DNA vector in accordance with conventional techniques, including blunt-ending or staggered-ending termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Maniatis, T., (Maniatis, T. et al., Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, second edition, 1988) and are well known in the art.
Libraries containing sequences coding for the desired gene may be screened and the desired gene sequence identified by any means which specifically selects for a sequence coding for such gene or protein such as, for example, a) by hybridization with an appropriate nucleic acid probe(s) containing a sequence specific for the DNA of this protein, or b) by hybridization-selected translational analysis in which native mRNA which hybridizes to the clone in question is translated in vitro and the translation products are further characterized, or, c) if the cloned genetic sequences are themselves capable of expressing mRNA, by immunoprecipitation of a translated protein product produced by the host containing the clone.
Oligonucleotide probes specific for a certain protein which can be used to identify clones to this protein can be designed from the knowledge of the amino acid sequence of the protein or from the knowledge of the nucleic acid sequence of the DNA encoding such protein or a related protein. Alternatively, antibodies may be raised against purified forms of the protein and used to identify the presence of unique protein determinants in transformants that express the desired cloned protein. When an amino acid sequence is listed horizontally, unless otherwise stated, the amino terminus is intended to be on the left end and the carboxy terminus is intended to be at the right end. Similarly, unless otherwise stated or apparent from the context, a nucleic acid sequence is presented with the 5' end on the left.
Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid. Peptide fragments may be analyzed to identify sequences of amino acids that may be encoded by oligonucleotides having the lowest degree of degeneracy. This is preferably accomplished by identifying sequences that contain amino acids which are encoded by only a single codon.
Although occasionally an amino acid sequence may be encoded by only a single oligonucleotide sequence, frequently the amino acid sequence may be encoded by any of a set of similar oligonucleotides. Importantly, whereas all of the members of this set contain oligonucleotide sequences which are capable of encoding the same peptide fragment and, thus, potentially contain the same oligonucleotide sequence as the gene which encodes the peptide fragment, only one member of the set contains the nucleotide sequence that is identical to the exon coding sequence of the gene. Because this member is present within the set, and is capable of hybridizing to DNA even in the presence of the other members of the set, it is possible to employ the unfractionated set of oligonucleotides in the same manner in which one would employ a single oligonucleotide to clone the gene that encodes the peptide.
Using the genetic code, one or more different oligonucleotides can be identified from the amino acid sequence, each of which would be capable of encoding the desired protein. The probability that a particular oligonucleotide will, in fact, constitute the actual protein encoding sequence can be estimated by considering abnormal base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic cells. Using "codon usage rules," a single oligonucleotide sequence, or a set of oligonucleotide sequences, that contain a theoretical "most probable" nucleotide sequence capable of encoding the protein sequences is identified.
The suitable oligonucleotide, or set of oligonucleotides, which is capable of encoding a fragment of a certain gene (or which is complementary to such an oligonucleotide, or set of oligonucleotides) may be synthesized by means well known in the art (see, for example, Oligonucleotides and Analogues, A Practical Approach, F. Eckstein, ed., 1992, IRL Press, New York) and employed as a probe to identify and isolate a clone to such gene by techniques known in the art. Techniques of nucleic acid hybridization and clone identification are disclosed by Maniatis, T., et al., in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y. (1982)), and by Hames, B. D., et al., in: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985)). Those members of the above-described gene bank which are found to be capable of such hybridization are then analyzed to determine the extent and nature of coding sequences which they contain.
To facilitate the detection of a desired DNA coding sequence, the above-described DNA probe is labeled with a detectable group. Such detectable group can be any material having a detectable physical or chemical property. Such materials have been well-developed in the field of nucleic acid hybridization and in general most any label useful in such methods can be applied to the present invention. Particularly useful are radioactive labels, such as 32 p, 3 H, 14 C, 35 S, 125 I, or the like. Any radioactive label may be employed which provides for an adequate signal and has a sufficient half-life. If single stranded, the oligonucleotide may be radioactively labelled using kinase reactions. Alternatively, polynucleotides are also useful as nucleic acid hybridization probes when labeled with a non-radioactive marker such as biotin, an enzyme or a fluorescent group.
Thus, in summary, the elucidation of a partial protein sequence, permits the identification of a theoretical "most probable" DNA sequence, or a set of such sequences, capable of encoding such a peptide. By constructing an oligonucleotide complementary to this theoretical sequence (or by constructing a set of oligonucleotides complementary to the set of "most probable" oligonucleotides), one obtains a DNA molecule (or set of DNA molecules), capable of functioning as a probe(s) for the identification and isolation of clones containing a gene.
In an alternative way of cloning a gene, a bank is prepared using an expression vector, by cloning DNA or, more preferably cDNA prepared from a cell capable of expressing the protein into an expression vector. The bank is then screened for members which express the desired protein, for example, by screening the bank with antibodies to the protein.
The above discussed methods are, therefore, capable of identifying genetic sequences that are capable of encoding a protein or biologically active or antigenic fragments of this protein. The desired coding sequence may be further characterized by demonstrating its ability to encode a protein having the ability to bind antibody in a specific manner, the ability to elicit the production of antibody which are capable of binding to the native, non-recombinant protein, the ability to provide a enzymatic activity to a cell that is a property of the protein, and the ability to provide a non-enzymatic (but specific) function to a recipient cell, among others.
In order to produce the recombinant protein in the vectors of the invention, it is desirable to operably link such coding sequences to the glucose regulatable promoters of the invention. When the coding sequence and the operably linked promoter of the invention are introduced into a recipient eukaryotic cell (preferably a fungal host cell) as a non-replicating DNA (or RNA), non-integrating molecule, the expression of the encoded protein may occur through the transient (nonstable) expression of the introduced sequence.
Preferably the coding sequence is introduced on a DNA molecule, such as a closed circular or linear molecule that is incapable of autonomous replication, Preferably, a linear molecule that integrates into the host chromosome. Genetically stable transformants may be constructed with vector systems, or transformation systems, whereby a desired DNA is integrated into the host chromosome. Such integration may occur de novo within the cell or, be assisted by transformation with a vector which functionally inserts itself into the host chromosome.
The gene encoding the desired protein operably linked to the promoter of the invention may be placed with a transformation marker gene in one plasmid construction and introduced into the host cells by transformation, or, the marker gene may be on a separate construct for co-transformation with the coding sequence construct into the host cell. The nature of the vector will depend on the host organism. In the practical realization of the invention the filamentous fungus Trichoderma has been employed as a model. Thus, for Trichoderma and especially for T. reesei, vectors incorporating DNA that provides for integration of the expression cassette (the coding sequence operably linked to its transcriptional and translational regulatory elements) into the host's chromosome are preferred. It is not necessary to target the chromosomal insertion to a specific site. However, targeting the integration to a specific locus may be achieved by providing specific coding or flanking sequences on the recombinant construct, in an amount sufficient to direct integration to this locus at a relevant frequency.
Cells that have stably integrated the introduced DNA into their chromosomes are selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector in the chromosome, for example the marker may provide biocide resistance, e.g., resistance to antibiotics, or heavy metals, such as copper, or the like. The selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transformation. A genetic marker especially for the transformation of the hosts of the invention is amdS, encoding acetamidase and thus enabling Trichoderma to grow on acetamide as the only nitrogen source. Selectable markers for use in transforming filamentous fungi include, for example, acetamidase (the amdS gene), benomyl resistance, oligomycin resistance, hygromycin resistance, aminoglycoside resistance, bleomycin resistance; and, with auxotrophic mutants, ornithine carbamoyltransferase (OCTase or the argB gene). The use of such markers is also reviewed in Finkelstein, D. B. in: Biotechnology of Filamentous Fungi: Technology and Products, Chapter 6, Finkelstein, D. B. et al., eds., Butterworth-Heinemann, publishers, Stoneham, Mass., (1992), pp. 113-156).
To express a desired protein and/or its active derivatives, transcriptional and translational signals recognizable by an appropriate host are necessary. The cloned coding sequences, obtained through the methods described above, and preferably in a double-stranded form, may be operably linked to sequences controlling transcriptional expression in an expression vector, and introduced into a host cell, either prokaryote or eukaryote, to produce recombinant protein or a functional derivative thereof. Depending upon which strand of the coding sequence is operably linked to the sequences controlling transcriptional expression, it is also possible to express antisense RNA or a functional derivative thereof.
Expression of the protein in different hosts may result in different post-translational modifications which may alter the properties of the protein. Preferably, the present invention encompasses the expression of the protein or a functional derivative thereof, in eukaryotic cells, and especially in fungus.
A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains expression control sequences which contain transcriptional regulatory information and such sequences are "operably linked" to the nucleotide sequence which encodes the polypeptide.
An operable linkage is a linkage in which a sequence is connected to a regulatory sequence (or sequences) in such a way as to place expression of the sequence under the influence or control of the regulatory sequence. Two DNA sequences (such as a coding sequence and a promoter region sequence linked to the 5' end of the coding sequence) are said to be operably linked if induction of promoter function results in the transcription of mRNA encoding the desired protein and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the expression regulatory sequences to direct the expression of the protein, antisense RNA, or (3) interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably linked to a DNA sequence if the promoter was capable of effecting transcription of that DNA sequence.
The precise nature of the regulatory regions needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribing and 5' non-translating (non-coding) sequences involved with initiation of transcription and translation respectively, such as the TATA box, capping sequence, CAAT sequence, and the like, with those elements necessary for the promoter sequence being provided by the promoters of the invention. Such transcriptional control sequences may also include enhancer sequences or upstream activator sequences, as desired.
Expression of a protein in eukaryotic hosts such as fungus requires the use of regulatory regions functional in such hosts, and preferably fungal regulatory systems. A wide variety of transcriptional and translational regulatory sequences can be employed, depending upon the nature of the host. Preferably, these regulatory signals are associated in their native state with a particular gene which is capable of a high level of expression in the host cell.
In eukaryotes, where transcription is not linked to translation, such control regions may or may not provide an initiator methionine (AUG) codon, depending on whether the cloned sequence contains such a methionine. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis in the host cell. Promoters from filamentous fungal genes which encode a mRNA product capable of translation are preferred, and especially, strong promoters can be employed provided they also function as promoters in the host cell.
As is widely known, translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine. For this reason, it is preferable to ensure that the linkage between a eukaryotic promoter and a DNA sequence which encodes the desired protein, or a functional derivative thereof, does not contain any intervening codons which are capable of encoding a methionine. The presence of such codons results either in a formation of a fusion protein (if the AUG codon is in the same reading frame as the protein-coding DNA sequence) or a frame-shift mutation (if the AUG codon is not in the same reading frame as the protein-coding sequence).
It may be desired to construct a fusion product that contains a partial coding sequence (usually at the amino terminal end) of a protein and a second coding sequence (partial or complete) of a second protein. The first coding sequence may or may not function as a signal sequence for secretion of the protein from the host cell. For example, the sequence coding for desired protein may be linked to a signal sequence which will allow secretion of the protein from, or the compartmentalization of the protein in, a particular host. Such fusion protein sequences may be designed with or without specific protease sites such that a desired peptide sequence is amenable to subsequent removal. In a preferred embodiment, the native signal sequence of a fungal protein is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the peptide that is operably linked to it. Aspergillus leader/secretion signal elements also function in Trichoderma.
If desired, the non-transcribed and/or non-translated regions 3' to the sequence coding for a desired protein can be obtained by the above-described cloning methods. The 3'-non-transcribed region may be retained for its transcriptional termination regulatory sequence elements, or for those elements which direct polyadenylation in eukaryotic cells. Where the native expression control sequences signals do not function satisfactorily in a host cell, then sequences functional in the host cell may be substituted.
The vectors of the invention may further comprise other operably linked regulatory elements such as DNA elements which confer antibiotic resistance, or origins of replication for maintenance of the vector in one or more host cells.
In another embodiment, especially for maintenance of the vectors of the invention in prokaryotic cells, or in yeast S. cerevisiae cells, the introduced sequence is incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. In Bacillus hosts, integration of the desired DNA may be necessary.
Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
When it is desired to use S. cerevisiae as a host for a shuttle vector, preferred S. cerevisiae yeast plasmids include those containing the 2-micron circle, etc., or their derivatives. Such plasmids are well known in the art (Botstein, D., et al., Miami Wntr. Symp. 19:265-274 (1982); Broach, J. R., in: The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., p. 445-470 (1981); Broach, J. R., Cell 28:203-204 (1982); Bollon, D. P., et al., J. Clin. Hematol. Oncol. 10:39-48 (1980); Maniatis, T., In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene Expression, Academic Press, NY, pp. 563-608 (1980)), and are commercially available.
Plasmid pMLO16 was deposited in an Eschericia coli host with the Deutsche Sammlung vonMikroorganismen und Zelkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124, F.R.G., on Jan. 27, 1998 (DSMZ accession number DSM 11963).
Plasmid pMLO16de15(11) was deposited in an Eschericia coli host with the Deutsche Sammlung vonMikroorganismen und Zelkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38 124, F.R.G., on Jan. 27, 1998 (DSMZ accession number DSM 11962).
Plasmid pMI-24 was deposited in an Eschericia coli host with the Deutsche Sammlung vonMikroorganismen und Zelkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124, F.R.G., on Jan. 27, 1998 (DSMZ accession number DSM 11964).
Plasmid pMI-27 was deposited in an Eschericia coli host with the Deutsche Sammlung vonMikroorganismen und Zelkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124, F.R.G., on Jan. 27, 1998 (DSMZ accession number DSM 11965).
Plasmid pMI-28 was deposited in an Eschericia coli host with the Deutsche Sammlung vonMikroorganismen und Zelkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124, F.R.G., on Jan. 27, 1998 (DSMZ accession number DSM 11966).
Once the vector or DNA sequence containing the construct(s) is prepared for expression, the DNA construct(s) is introduced into an appropriate host cell by any of a variety of suitable means, including transformation. After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. If this medium includes glucose, expression of the cloned gene sequence(s) results in the production of the desired protein, or in the production of a fragment of this protein as desired. This expression can take place in a continuous manner in the transformed cells, or in a controlled manner, for example, by induction of expression.
Fungal transformation is carried out also accordingly to techniques known in the art, for example, using, for example, homologous recombination to stably insert a gene into the fungal host and/or to destroy the ability of the host cell to express a certain protein.
Fungi useful as recombinant hosts for the purpose of the invention include, e.g. Trichoderma, Aspergillus, Claviceps purpurea, Penicillium chrysogenum, Magnaporthe grisea, Neurospora, Mycosphaerella spp., Collectotrichum trifolii, the dimorphic fungus Histoplasmia capsulatum, Nectria haematococca (anamorph:Fusarium solani f. sp. phaseoli and f. sp. pisi), Ustilago violacea, Ustilago maydis, Cephalosporium acremonium, Schizophyllum commune, Podospora anserina, Sordaria macrospora, Mucor circinelloides, and Collectotrichum capsici. Transformation and selection techniques for each of these fungi have been described (reviewed in Finkelstein, D. B. in: Biotechnology of Filamentous Fungi: Technology and Products, Chapter 6, Finkelstein, D. B. et al., eds., Butterworth-Heinemann, publishers, Stoneham, Mass., (1992), pp. 113-156). Especially preferred are Trichoderma reesei, T. harzianum, T. longibrachiatum, T. viride, T. koningii, Aspergillus nidulans, A. niger, A. terreus, A. ficum, A. oryzae, A. awamori and Neurospora crassa.
The hosts of the invention are meant to include all Trichoderma. Trichoderma are classified on the basis of morphological evidence of similarity. T. reesei was formerly known as T. viride Pers. or T. koningii Oudem; sometimes it was classified as a distinct species of the T. longibrachiatum group. The entire genus Trichoderma, in general, is characterized by rapidly growing colonies bearing tufted or pustulate, repeatedly branched conidiophores with lageniform phialides and hyaline or green conidia borne in slimy heads (Bissett, J., Can. J. Bot. 62:924-931 (1984)).
The fungus called T. reesei is clearly defined as a genetic family originating from the strain QM6a, that is, a family of strains possessing a common genetic background originating from a single nucleus of the particular isolate QM6a. Only those strains are called T. reesei.
Classification by morphological means is problematic and the first recently published molecular data from DNA-fingerprint analysis and the hybridization pattern of the cellobiohydrolase 2 (cbh2) gene in T. reesei and T. longibrachiatum clearly indicates a differentiation of these strains (Meyer, W. et al., Curr. Genet. 21:27-30 (1992); Morawetz, R. et al., Curr. Genet. 21:31-36 (1992).
However, there is evidence of similarity between different Trichoderma species at the molecular level that is found in the conservation of nucleic acid and amino acid sequences of macromolecular entities shared by the various Trichoderma species. For example, Cheng, C., et al., Nucl. Acids. Res. 18:5559 (1990), discloses the nucleotide sequence of T. viride cbh1. The gene was isolated using a probe based on the T. reesei sequence. The authors note that there is a 95% homology between the amino acid sequences of the T. viride and T. reesei gene. Goldman, G. H. et al., Nucl. Acids Res. 18:6717 (1990), discloses the nucleotide sequence of phosphoglycerate kinases from T. Viride and notes that the deduced amino acid sequence is 81% homologous with the phosphoglycerate kinase gene from T. reesei . Thus, the species classified to T. viride and T. reesei must genetically be very close to each other.
In addition, there is a high similarity of transformation conditions among the Trichoderma. Although practically all the industrially important species of Trichoderma can be found in the formerly discussed Trichoderma section Longbrachiatum, there are some other species of Trichoderma that are not assigned to this section. Such a species is, for example, Trichoderma harzianum, which acts as a biocontrol agent against plant pathogens. A transformation system has also been developed for this Trichoderma species (Herrera-Estrella, A. et al., Molec. Microbiol. 4:839-843 (1990)) that is essentially the same as that taught in the application. Thus, even though Trichoderma harzianum is not assigned to the section Longibrachiatum, the method used by Herrera-Estrella in the preparation of spheroplasts before transformation is the same. The teachings of Herrera-Estrella show that there is not a significant diversity of Trichoderma spp. such that the transformation system of the invention would not be expected to function in all Trichoderma.
Further, there is a common functionality of fungal transcriptional control signals among fungal species. At least three A. nidulans promoter sequences, amdS, argB, and gpd, have been shown to give rise to gene expression in T. reesei . For amdS and argB, only one or two copies of the gene are sufficient to being about a selectable phenotypes (Penttila et al., Gene 61:155-164(1987)). Gruber, F. et al., Curr. Genetic 18:71-76 (1990) also notes that fungal genes can often by successfully expressed across different species. Therefore, it is to be expected that the glucose regulated promoters identified herein would be also regulatable by glucose in other fungi.
Many species of fungi, and especially Trichoderma, are available from a wide variety of resource centers that contain fungal culture collections. In addition, Trichoderma species are catalogued in various databases. These resources and databases are summarized by O'Donnell, K. et al., in Biochemistry of Filamentous Fungi: Technology and Products, D. B. Fingelstein et al., eds., Butterworth-Heinemann, Stoneham, Mass., USA, 1992, pp. 3-39.
After the introduction of the vector and selection of the transformant, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the synthesis and secretion of the desired heterologous or homologous protein, or in the production of a fragment of this protein, into the medium of the host cell.
In a preferred embodiment, the coding sequence is the sequence of an enzyme that is capable of hydrolysing lignocellulose. Examples of such sequences include a DNA sequence encoding cellobiohydrolase I (CBHI), cellobiohydrolase II (CBHII), endoglucanase I (EGI), endoglucanase II (EGII), endoglucanase III (EGIII), β-glucosidases, xylanases (including endoxylanases and β-xylosidase), side-group cleaving activities, (for example, α-arabinosidase, α-D-glucuronidase, and acetyl esterase), mannanases, pectinases (for example, endo-polygalacturonase, exo-polygalacturonase, pectinesterase, or, pectin and pectin acid lyase), and enzymes of lignin polymer degradation, (for example, lignin peroxidase LIII from Phlebia radiata (Saloheimo et al., Gene 85:343-351 (1989)), or the gene for another ligninase, lacease or Mn peroxidase (Kirk, In: Biochemistry and Genetics of Cellulose Degradation, Aubert et al. (eds.), FEMS Symposium No. 43, Academic Press, Harcourt, Brace Jovanovitch Publishers, London. pp. 315-332 (1988))). The cloning of the cellulolytic enzyme genes has been described and recently reviewed (Teeri, T. T. in: Biotechnology of Filamentous Fungi: Technology and Products, Chapter 14, Finkelstein, D. B. et al., eds., Butterworth-Heinemann, publishers, Stoneham, Mass., (1992), pp. 417-445). The gene for the native cellobiohydrolase CBHI sequence has been cloned by Shoemaker et al. (Shoemaker, S., et al., Bio/Technology 1:691-696 (1983)) and Teeri et al. (Teeri, T., et al., Bio/Technology 1:696-699 (1983)) and the entire nucleotide sequence of the gene is known (Shoemaker, S., et al., Bio/Technology 1:691-696 (1983)). From T. reesei, the gene for the major endoglucanase (EGI) has also been cloned and characterized (Penttila, M., et al., Gene 45:253-263 (1986); Patent Application EP 137,280; Van Arstel, J. N. V., et al., Bio/Technology 5:60-64). Other isolated cellulase genes include cbh2 (Patent Application WO 85/04672; Chen, C. M., et al., Bio/Technology 5:274-278 (1987)) and egl3 (Saloheimo, M., et al., Gene 63:11-21 (1988)). The genes for the two endo-p-xylanases of T. reesei (xln1 and xln2) have been cloned and described in applicants'copending application, U.S. Ser. No. 07/889,893, filed May 29, 1992. The xylanase proteins have been purified and characterized (Tenkanen, M. et al., Proceeding of the Xylans and Xylanases Symposium, Wageningen, Holland (1991)).
The expressed protein may be isolated and purified from the medium of the host in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like. For example, the cells may be collected by centrifugation, or with suitable buffers, lysed, and the protein isolated by column chromatography, for example, on DEAE-cellulose, phosphocellulose, polyribocytidylic acid-agarose, hydroxyapatite or by electrophoresis or immunoprecipitation.
The manner and method of carrying out the present invention may be more fully understood by those of skill by reference to the following examples, which examples are not intended in any manner to limit the scope of the present invention or of the claims directed thereto.
Isolation of Trichoderma reesei Genes Strongly Expressed on Glucose
For the isolation of glucose induced mRNA Trichoderma reesei strain QM9414 (Mandels, M. et al., Appl. Microbiol. 21:152-154 (1971)) was grown in a 10 liter fermenter in glucose medium (glucose 60 g/l, Bacto-Peptone 5 g/l, Yeast extract 1 g/l, KH2 PO4 4 g/l, (NH4)2 SO4 4 g/l, MgSO4 0.5 g/l, CaCl2 0.5 g/l and trace elements FeSO4.7H2 O 5 mg/l, MnSO4.H2 O 1.6 mg/l, ZnSO4.7H2 O 1.4 mg/l, and CoCl2.6H2 O 3.7 mg/l, pH 5.0-4.0). Glucose feeding (465 g/20 h) was started after 30 hours of growth. Mycelium was harvested at 45 hours of growth and RNA was isolated according to Chirgwin, J. M. et al., Biochem. J. 18:5294-5299 (1979). Poly A+ RNA was isolated from the total RNA by oligo(dT)-cellulose chromatography (Maniatis, T. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982)) and cDNA synthesis and cloning of the cDNAs was carried out according to manufacturer's instructions into lambda-ZAP vector (ZAP-cDNA synthesis kit, Stratagene). The cDNA bank was transferred onto nitrocellulose filters and screened with 32 P-labelled single-stranded cDNA synthesized (Teeri, T. T. et al., Anal. Biochem. 164:60-67 (1987)) from the same poly A+ RNA from which the bank was constructed. The labelled cDNA was relabelled with 32 P-dCTP (Random Primed DNA Labeling kit, Boehringer-Mannheim). The hybridization conditions were as described in Maniatis, T. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). Fifty clones giving the strongest positive reaction were isolated and the cDNAs were subcloned in vivo into Bluescript SK(-) plasmid according to manufacturer's instructions (ZAP-cDNA synthesis kit, Stratagene).
To identify the clones and exclude the same ones they were all sequenced from the 3' end by using standard methods. The frequency of each specific clone in the cDNA lambda-bank was determined by hybridizing the bank with a clone specific PCR probe. The clones cDNA33, cDNA1, cDNA10, cDNA12, cDNA15, showing the five highest frequencies corresponded to 1-3% of the total mRNA pool.
Characterization of Isolated Glucose Expressed Trichoderma Genes and Their Promoters
The cDNAs of the clones cDNA33, cDNA1, cDNA10, cDNA12, and cDNA15 were used as probes to isolate the corresponding genes and promoters from a Trichoderma chromosomal lambda-bank prepared earlier (Vanhanen, S. et al., Curr. Genet. 15:181-186 (1989)). On the basis of Southern analysis of restriction enzyme digestions carried out for the chromosomal lambda clones, the promoters and either the 5' parts of the chromosomal genes or the whole genes were subcloned into pSP73 vector (Promega, Madison, USA) using appropriate restriction enzymes yielding the plasmids pTHN1 (FIG. 1), pEA33 (FIG. 2), pTHN3 (FIG. 3), pEA10 (FIG. 4), pEA12 (FIG. 5) and pEA155 (FIG. 6), corresponding to the clones cDNA33, cDNA1, cDNA10, cDNA12 and cDNA15, respectively. Sequences were obtained from the 5' ends of the genes and from the promoters using primers designed from previously obtained sequences. The sequences of the isolated promoters and genes or parts of them (either obtained from cDNA or chromosomal DNA) are shown in SEQ ID1 for cDNA33, SEQ ID2 for cDNA1, SEQ ID3 for cDNA10, SEQ ID4 for cDNA12, and SEQ ID5 for cDNA15. Based on sequence similarity to known sequences in a protein data bank the clone cDNA33 could be identified as a translation elongation factor, TEF1α.
Construction of Vectors for Expression of EGI-core under the tef1-Promoter in Trichoderma
A XhoI+DraIII fragment that is internal to the egl1 cDNA [SEQ ID 16 and FIG. 7B] sequence of plasmid pPLE3 (FIG. 7) carrying the EcoRI-BamHI fragment of egl1 cDNA from pTTc11 (Penttila et al., Gene 45:253-263 (1986); Penttila et al., Yeast 3:175-185 (1987)) inbetween the cbh1 promoter and c. 700 nt long AvaII terminator fragment was replaced by a XhoI-DraIII fragment of cDNA from plasmid pEG131 (Nitisinprasert, S., Reports from Department of Microbiology, University of Helsinki (1990)). The pPEG131 insert sequence is egl1 cDNA in which a STOP codon is constructed just before the hinge region of the egl1 gene. The cbh1 terminator sequence is FIG. 7C [SEQ ID 23]. SEQ ID 23 is a shortened cbh1 terminator sequence, similar to SEQ ID 24 (the "long" cbh1 terminator but lacking 30 nucleotides at the 5' end).
pPLE3 contains a pUC18 backbone, and carries the cbh1 promoter inserted at the EcoRI site. The cbh1 promoter is operably linked to the full length egl1 cDNA coding sequence and to the cbh1 transcriptional terminator. The ori and amp genes are from the bacterial plasmid.
The resulting plasmid pEM-3 (FIG. 8) now carries a copy of egl1 cDNA with a translational stop codon after the egl1 core region (EGI amino acids 1-22 are the EGI signal sequence; EGI amino acids 23-393, terminating at a Thr, are considered the `core` sequence). pEM-3 was then digested with EcoRI and SphI and the released Bluescribe M13+ moiety (Vector Cloning Systems, San Diego, USA) of the plasmid was replaced by EcoRI and SphI digested pAMD (FIG. 8) containing a 3.4 kb amdS fragment from plasmid p3SR2 (Hynes, M. J. et al., Mol. Cell. Biol. 3:1430-1439 (1983); Tilburn, J. et al., Gene 26:205-221 (1983). This resulting plasmid pEM-3A (FIG. 8) was digested with EcoRI and partially with KspI to release the 2.3 kb fragment carrying the cbh1-promotor and the 8.6 kb fragment carrying the rest of the plasmid was purified from agarose gel. Based on the sequence data of the tef1 promoter (SEQ ID1 bases 1-1234), two primers were designed (SEQ ID6 and SEQ ID7) and used in a PCR reaction to isolate a 1.2 kb promoter fragment adjacent to the translational start site of the tef1 gene. The, 5' primer was ACCGGAATTCATATCTAGAGGAGCCCGCGAGTTTGGATACGCC (SEQ ID NO:6) and the 3' primer was ACCGCCGCGGTTTGACGGTTTGTGTGATGTAGCG (SEQ ID NO:7). The bold and underlined GAATTC in the 5' primer is an EcoRI site. The bold and underlined TCTAGA in the 5' primer is an XbaI site. The bold and underlined CCGCGG in the 3' primer is a SacII site. This fragment was digested with EcoRI and partially with KspI and purified from agarose gel and ligated to the 8.6 kb pEM-3A fragment resulting in plasmid pTHN100B (FIG. 9). This expression vector carries DNA encoding the EGI-core construction operably linked to the tef1 promoter; this plasmid also carries an amdS marker gene for selection of Trichoderma transformants.
Transformation of Trichoderma, Purification of the EGI-Core Producing Clones and Their Analysis
Trichoderma reesei strain QM9414 was transformed essentially as described (Penttila, M. et al., Gene 61:155-164 (1987) using 6-10 μg of the plasmid pTHN100B. The Amd+ transformants obtained were streaked twice onto slants containing acetamide (Penttila, M. et al. Gene 61:155-164 (1987)). Thereafter spore suspensions were made from transformants grown on Potato Dextrose agar (Difco). EGI-core production was tested by slot blotting with EGI specific antibody from 50 ml shake flask cultures carried out in minimal medium (Penttila, M. et al. Gene 61:155-164 (1987)) supplemented with 5% glucose and using additional glucose feeding (total amount of fed glucose was 6 ml of 20% glucose). The spore suspensions of the EGI-core producing clones were purified to single spore cultures on Potato Dextrose agar plates. EGI-core production was analyzed again from these purified clones as described above (FIG. 10).
Characterization of EGI-core produced by Trichoderma Grown on Glucose
EGI-core producing strain pTHN100B-16c was grown in a 10 liter fermenter in glucose medium as described earlier in Example 1 except that yeast extract was left out and glucose feeding was 555 g/22 h. The culture supernatant was separated from the mycelium by centrifugation. The secretion of EGI-core by Trichoderma was verified by Western blotting by conventional methods running concentrated culture supernatants on SDS-PAGE and treating the blotted filter with monoclonal EGI-core specific antibodies (FIG. 11 and FIG. 12). The enzyme activity was shown semiquantitatively in a microtiter plate assay by using the concentrated culture supernatants and 3 mM chloronitrophenyl lactocide as a substrate and measuring the absorbance at 405 nm (Clayessens, M. et al., Biochem. J. 261:819-825 (1989)).
Construction of β-Galactosidase Expression Vectors with Truncated Fragments of the cbh1-Promoter
The vector pMLO16 (FIG. 13A) contains a 2.3 kb cbh1 promoter fragment ([SEQ ID NO:18, FIGS. 13B and 13C) starting at 5' end from the EcoRI site, isolated from chromosomal gene bank of Trichoderma reesei (Teeri, T. et al., Bio/Technology 1:696-699 (1983)), a 3.1 kb BamHI fragment of the lacZ gene from plasmid pAN924-21 (van Gorcom et al., Gene 40:99-106 (1985)) and a 1.6 kb cbh1 terminator (FIGS. 13D and 13E, [SEQ ID NO:24]) starting from 84 bp upstream from the translation stop codon and extending to a BamHI site at the 3' end (Shoemaker, S. et al., Bio/Technology 1:691-696 (1983); Teeri, T. et al., Bio/Technology 1:696-699 (1983)). These pieces were linked to a 2.3 kb long EcoRI-PvuII region of pBR322 (Sutcliffe, J. G., Cold Spring Harbor Symp. Quant. Biol. 43:77-90 (1979)) generating junctions as shown in FIG. 13. The exact in frame joint between the 2.3 kb cbh1 promoter and the 3.1 kb lacZ gene was constructed by using an oligo depicted in FIG. 13A. A polylinker shown in FIG. 13A was cloned into the single internal XbaI site in the cbh1 promoter for the purpose of promoter deletions. A short SalI linker shown in FIG. 13A was cloned into the joint between the pBR322 and cbh1 promoter fragments so that the expression cassette can be released from the vector by restriction digestion with SalI and SphI. Progressive unidirectional deletions were introduced to the cbh1 promoter by cutting the vector with KpnI and XhoI and using the Erase-A-Base System (Promega, Madison, USA) according to manufacturer's instructions. Plasmids obtained from different deletion time points were transformed into the E. coli strain DH5α (BRL) by the method described in (Hanahan D., J. Mol. Biol. 166:557-580 (1983)) and the deletion end points were sequenced by using standard methods.
Transformation of Trichoderma, Isolation of the β-Galactosidase Producing Clones and Their Analysis
Trichoderma reesei strain QM9414 was transformed with expression vectors for β-galactosidase containing either the intact 2.3 kb cbh1 promoter or truncated versions of it, generated as explained in Example 6. Twenty μg of the plasmids were digested with SalI and SphI to release the expression cassettes from the vectors and these mixtures were cotransformed to Trichoderma together with 3 μg of plasmid p3SR2 (Hynes, M. J. et al., Mol. Cell. Biol. 3:1430-1439 (1983)) containing the acetamidase gene. The transformation method was that described in (Penttila, M. et al. Gene 61:155-164 (1987)) and the Amd+ transformants were screened as described earlier in Example 4. The β-galactosidase production of the Amd+ transformants was tested by inoculating spore suspensions on microtiter plate wells containing solid minimal medium (Penttila, M. et al. Gene 61:155-164 (1987)) supplemented with 2% glucose, 2% fructose and 0.2% peptone and pH adjusted to 7. After 24 h incubation in 28°C, 10 μl of the chromogenic substrate X-gal (20 mg/ml) was added to each well and the formation of blue color was followed as an indication of β-galactosidase activity. An intense blue color could be detected in transformants transformed with a plasmid pMLO16de15(11) (FIG. 14) containing a 1110 bp deletion in the cbh1 promoter beginning from the promoter internal polylinker and ending 385 bp before the translation initiation site (FIG. 15). The sequence of this truncated promoter is provided as SEQ ID19 (FIG. 15B).
Production of CBHI on Glucose with the Glucose-Derepressed cbh1-Promoter
For the production of CBHI on glucose an expression plasmid pMLO 17 (FIG. 16) was constructed. The plasmid pMLO16de15(11) was digested with the enzymes KspI (the first nucleotide of the recognition sequence is at the position -16 from the ATG) and XmaI (the first nucleotide of the recognition sequence is 76 nucleotides downstream from the translation stop codon of the cbh1 gene). The vector part containing the shortened cbh1 promoter, the cbh1 terminator and the pBR322 sequence was ligated to the chromosomal cbh1 gene isolated as a KspI-XmaI-fragment from the chromosomal gene bank of Trichoderma reesei (Teeri, T. et al., Bio/Technology 1:696-699 (1983)). The sequence of this fragment is provided as the underlined portion of FIGS. 16B and 16C ([SEQ ID NO:17]). The plasmid pMLO17 was transformed to the Trichoderma reesei strain QM 9414 and the Amd+ transformants were screened as described earlier in example 7. CBHI production was tested from 40 transformants in microtiter plate cultures (200 μl; 3 days) carried out in minimal medium (Penttila, M. et al. Gene 61:155-164 (1987) supplemented with 3% glucose and using additional glucose feeding (total amount of fed glucose was 6 mg/200 μl culture). The culture supernatants were slot blotted on nitrocellulose filters and CBHI was detected with specific antibody. The spore suspensions of the 10 best CBHI producing transformants were purified to single spore cultures on plates containing acetamide and Triton X-100 (Penttila, M. et al., Gene 61:155-164 (1987)). Thirty single spore cultures were tested for CBHI production in shake flask cultivations (50 ml; 6 days) carried out in the same medium as described above. The total amount of fed glucose was 1.8 g/50 ml culture. Dilutions of the culture supernatants were slot blotted and CBHI was detected with specific antibody (FIGS. 17A and 17B).
β-Galactosidase Expression Vectors with Specific Mutations in cbh1 Promoter to Release Glucose Repression
Six 6 bp sequences found in cbh1 promoter similar to binding sites of Saccharomyces cerevisiae glucose repressor protein MIG1 (Nehlin & Ronne, EMBO J. 9:2891-2899 (1990); Nehlin et al., EMBO J. 10:3373-3377 (1991)) were changed into other nucleotides to study the functionality of these mig-like sequences in mediating the glucose repression of the native cbh1 promoter of Trichoderma reesei. To construct β-galactosidase expression vectors with cbh1 promoters carrying specific mutations, sequence alterations were made into primers (specifically: TCT TCA AGA ATT GCT CGA CCA ATT CTC ACG GTG AAT GTA GG (SEQ ID NO:8); ACA CAT CTA GAG GTG ACC TAG GCA TTC TGG CCA CTA GAT ATA TAT TTA GAA GGT TCT TGT AGC TCA AAA GAG C (SEQ ID NO:9): GGG AAT TCT CTA GAA ACG CGT TGG CAA ATT ACG GTA CG (SEQ ID NO:10); GGG AAT TCG GTC ACC TCT AAA TGT GTA ATT TGC CTG CTT GAC C (SEQ ID NO:11); GGG AAT TCG GTC ACC TCT AAA TGT GTA ATT TGC CTG CTT GAC CGA TCT AAA CTG TTC GAA GCC CGA ATG TAG G (SEQ ID NO:12); GGG AAT TCT TCT AGA TTG CAG AAG CAC GGC AAA GCC CAC TTA CCC (SEQ ID NO:13); TAG CGA ATT CTA GGT CAC CTC TAA AGG TAC CCT GCA GCT CGA GCT AG (SEQ ID NO:14); and GGG AAT TCA TGA TGC GCA GTC CGC GG (SEQ ID NO:15); these primers were specific for the cbh1 promoter and the cbh1 promoter internal polylinker and were used in PCR amplification of cbh1 promoter sequences for cloning.
pMLO16 (FIG. 13) was used as a PCR template with the appropriate primers to yield a 770 bp fragment A (primers TAG CGA ATT CTA GGT CAC CTC TAA AGG TAC CCT GCA GCT CGA GCT AG (SEQ ID NO:14) and GGG AAT TCT CTA GAA ACG CGT TGG CAA ATT ACG GTA CG (SEQ ID NO:10), beginning at the polylinker at -1497 and ending at -726 upstream of ATG, and a 720 bp fragment B (primers GGG AAT TCT TCT AGA TTG CAG AAG CAC GGC AAA GCC CAC TTA CCC (SEQ ID NO:13) and GGG AAT TCA TGA TGC GCA GTC CGC GG (SEQ ID NO:15)), beginning at -719 and ending at KspI at -16. Fragments A and B were purified from agarose gel and digested with BstEII-XbaI and XbaI-KspI respectively, ligated to the 7.8 kb fragment of pMLO16 to produce pMI-24. The cbh1 promoter of pMI-24 has sequence alterations at positions -725 to -720 (genomic sequence 5': GTGGGG, altered sequence: 5' TCTAGA)), -699 to -694 (genomic sequence: 5' CCCCAC, altered sequence: 5' CCCAC), and -691 to -686 (genomic sequence: 5' CCCCAC, altered sequence: 5' ACCCAC) upstream of the translation initiation codon of intact cbh1 promoter (FIG. 18A). The sequence of the altered cbh1 promoter in pMI-24 is provided in FIGS. 18B and 18C and SEQ ID NO:20.
pMLO16de10(2) (FIG. 19) containing a 460 bp deletion in the cbh1 promoter beginning from the promoter internal polylinker and ending 1025 bp before the translation initiation site was constructed as described in Example 6 and used as a PCR template with primers (TCT TCA AGA ATT GCT CGA CCA ATT CTC ACG GTG AAT GTA GG (SEQ ID NO:8) and ACA CAT CTA GAG GTG ACC TAG GCA TTC TGG CCA CTA GAT ATA TAT TTA GAA GGT TCT TGT AGC TCA AAA GAG C (SEQ ID NO:9)) to yield a 800 bp fragment C, beginning from the 5' end of cbh1 promoter and ending at the promoter internal polylinker. Fragment C was purified from agarose gel, digested with SalI-XbaI and ligated to the 7.6 kb SalI-XbaI fragment of pMLO16de10(2) to produce pMI-25. The cbh1 promoter of pMI-25 has a sequence alteration (genomic sequence: 5'GTGGGG, altered sequence: 5'TCTAAA) at position -1510 to 1505 upstream of the translation initiation codon of intact cbh1 promoter (FIG. 18).
pMLO16de10(2) was used as a PCR template to yield a 750 bp fragment D (primers GGG AAT TCG GTC ACC TCT AAA TGT GTA ATT TGC CTG CTT GAC CGA TCT AAA CTG TTC GAA GCC CGA ATG TAG G (SEQ ID NO:12) and GGG AAT TCA TGA TGC GCA GTC CGC GG (SEQ ID NO:15)), beginning from the promoter internal polylinker and ending at KspI at -16. Fragment D was purified from agarose gel, digested with BstEII-KspI and ligated to the 7.8 kb BstEII-KspI fragment of pMI-25 to produce pMI-26. The cbh1 promoter of pMI-26 has sequence alterations at positions -1510 to -1505 (genomic sequence: 5'GTGGGG, altered sequence: 5'TCTAAA) and -1006 to -1001 (genomic sequence: 5'CTGGGG, altered sequence: 5'CTAAA) upstream of the translation initiation codon of intact cbh1 promoter (FIG. 18).
pMLO16de10(2) was used as a PCR template to yield a 280 bp fragment E (primers GGG AAT TCT CTA GAA ACG CGT TGG CAA ATT ACG GTA CG (SEQ ID NO:10) and GGG AAT TCG GTC ACC TCT AAA TGT GTA ATT TGC CTG CTT GAC C (SEQ ID NO:11)), beginning from the promoter internal polylinker and ending at -720 and a 720 bp fragment F (primers GGG AAT TCT TCT AGA TTG CAG AAG CAC GGC AAA GCC CAC TTA CCC (SEQ ID NO:13) and GGG AAT TCA TGA TGC GCA GTC CGC GG (SEQ ID NO:15)), beginning at -720 and ending at KspI at -16. Fragments D and E were purified from agarose gel, digested with BstEII-XbaI and XbaI-KspI respectively and ligated to the 7.8 kb BstEII-KspI fragment of pMI-25 to produce pMI-27. The cbh1 promoter of pMI-27 has sequence alterations at positions -1510 to -1505 (genomic sequence: 5'GTGGGG, altered sequence: 5'CTAAA) -725 to -720 (genomic sequence 5' GTGGGG, altered sequence: 5' TCTAGA), -699 to -694 (genomic sequence: 5 CCCCAC. altered sequence: 5' CCCAC), and -691 to -686 (genomic sequence: 5' CCCCAC, altered sequence: 5' ACCCAC) upstream of the translation initiation codon of intact cbh1 promoter (FIG. 18). The sequence of the altered cbh1 promoter of pMI-27 is shown in FIGS. 18D and 18E and SEQ ID NO:21.
pMLO16de10(2) was used as a PCR template to yield a 280 bp fragment G (primers GGG AAT TCT CTA GAA ACG CGT TGG CAA ATT ACG GTA CG (SEQ ID NO:10) and GGG AAT TCG GTC ACC TCT AAA TGT GTA ATT TGC CTG CTT GAC CGA TCT AAA CTG TTC GAA GCC CGA ATG TAG G (SEQ ID NO:12)), beginning from the promoter internal polylinker and ending at -720 and a 720 bp fragment H (primers GGG AAT TCT TCT AGA TTG CAG AAG CAC GGC AAA GCC CAC TTA CCC (SEQ ID NO:13) and GGG AAT TCA TGA TGC GCA GTC CGC GG (SEQ ID NO:15)), beginning at -720 and ending at KspI at -16. Fragments G and H were purified from agarose gel, digested with BstEII-XbaI and XbaI-KspI respectively and ligated to the 7.8 kb BstEII-KspI fragment of pMI-25 to produce pMI-28. The cbh1 promoter of pMI-28 has sequence alterations at positions -1510 to -1505 (genomic sequence: 5'GTGGGG, altered sequence: 5'CTAAA), -1006 to -1001 (genomic sequence: 5' CTGGGG, altered sequence: 5'TCTAAA) -725 to -720 (genomic sequence 5' GTGGGG, altered sequence: 5' TCTAGA), -699 to -694 (genomic sequence: 5' CCCCAC, altered sequence: 5' CCCAC), and -691 to -686 (genomic sequence: 5' CCCCAC, altered sequence: 5' ACCCAC) upstream of the translation promoter (FIG. 18). The sequence of the altered cbh1 promoter of pMI-28 is shown in FIGS. 18F and 18G and SEQ ID NO:22.
All PCR amplified DNA fragments and ligation joints were sequenced using standard methods to ensure that the mutations were present and no other nucleotides were changed. Transformation of Trichoderma reesei QM9414 with the vectors mentioned above, isolation of β-galactosidase producing clones and their analysis was done as described in Example 7. After addition of X-gal, an intense blue color was detected on glucose grown transformant colonies as an indication of β-galactosidase activity in transformants transformed with the plasmids pMI-24, pMI-27 and pMI-28 (FIG. 20), indicating that altering the cbh1 promoter according to any of those mutations was sufficient to allow for expression of proteins in Trichoderma under the cbh1 promoter in the presence of glucose.
| __________________________________________________________________________ |
| # SEQUENCE LISTING |
| - - - - (1) GENERAL INFORMATION: |
| - - (iii) NUMBER OF SEQUENCES: 34 |
| - - - - (2) INFORMATION FOR SEQ ID NO:1: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 3461 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: |
| - - CGCCGTGACG ACAGAAACGG AGCCCGCGAG TTTGGATACG CCGCTGAAAT GG - |
| #GGCTTGAC 60 |
| - - GGTGAAGGAG AAGCCGAGCG CGGTGCCAGA GGACAAGATG GATGTAGAGC CA - |
| #GGCGACGA 120 |
| - - CGACCAAACG CAACCATCAA ATCAATCAGA TGGCAATGAC GCACCACCGC CC - |
| #CAGCAGCG 180 |
| - - CGAACCGCCG ACGAAGAAGC CATGGACGCG CTCCTCGGCA AGACGCCCAA GG - |
| #AACAGAAA 240 |
| - - AAAGTAATCT CCGCACCCGT ATCAGAAGAC GACGCCTACC GCCGCGACGT CG - |
| #AAGCCTCC 300 |
| - - GGCGCGGTGT CCACGCTCCA GGATTACGAA GACATGCCCG TCGAGGAGTT TG - |
| #GCGCCGCC 360 |
| - - CTCCTCCNNN GCATGGGCTG GAACGGGGAA GCCCGCGGCC CGCCGGTCAA GC - |
| #AGGTCAAG 420 |
| - - AGGCGGCAGA ACAGGCTCGG CCTCGGCGCC AAGGAGCTCA AGGAGGAAGA GG - |
| #ACCTCGGC 480 |
| - - GGGTGGAACC AGAACGGCAA GAAAAAGTCG AGGCCSCGCG GCTGAGCGAG TA - |
| #TCGGAGGG 540 |
| - - AGGAGAGCAA GCGCAAGGAA GGCCGGGGGC ATGAGGACAG CTATAAACGA GA - |
| #GAGGGAGC 600 |
| - - GCGAACGGAT CGCGAGAGGG ATCACTACAG GGAGCGAGAC CGGGACAGGG AT - |
| #CGCGATTA 660 |
| - - TAGGGATCGG GATAGGGATA GACATCGGGA CCACGATAGG CACAGGGACC GA - |
| #CATCGCGA 720 |
| - - CTCTGACCGG CACCATCGAC GATGAAGGAG CTTTTGCATT CTTCTCTTCG TC - |
| #AACCACTT 780 |
| - - TTGAGACTAA CATTAACCAT GCCGTTTTCT TGAAAAGCTT GTACTCATCA TG - |
| #ATGTTTTT 840 |
| - - AAGCAAATAG GCGACAGGCG TACAGACACC TTAATATCAC ATAGAGGCAC GG - |
| #CACACATA 900 |
| - - CGTCTTGGAG AAGACACGTA CTTACGAATG ATGGGAGAAT TACCTACTCT GA - |
| #CTTGTGTA 960 |
| - - AATTAGAATA TCAATGACAC TATGTATATT CAGTCGAGCT GCGAATGGTC AC - |
| #ACATTGTC 1020 |
| - - TGATCTGCGA ATTTGTATGT GCTGCCTCTC CCTCTGACCT TCTGGTCTGG TG - |
| #ATACCATC 1080 |
| - - CTCCCTCAGT TTGGATCATC GCCTTATTCT TCTTCCCTCT TCTGCATCTG CT - |
| #TCCTGCTC 1140 |
| - - GTTTGAGGAA CATCGCCAGC TGACTCTGCT TGCCTCGCAG CGATCTAGTC AA - |
| #GAACAACA 1200 |
| - - CNAGCTCTCA CGCTACATCA CACAAACCGT CAAAATGGGT AAGGAGGACA AG - |
| #ACTCACAT 1260 |
| - - CAACGTGGTC GTCATCGTAC GTATTTTCCG ATCCCTCATC GGCNGTCATC TG - |
| #NCCAGTCT 1320 |
| - - GATTCCAAGA ATCACCGTGC TAACCATATA CCATCTANGG GTGCGTATTC CA - |
| #TCAATCAT 1380 |
| - - CTTGAGCCAG ATCGACCGAA CATACGATAC TGACTTTGCT ACGACAGCCA CG - |
| #TCGACTCC 1440 |
| - - GGCAAGTCTA CCACCGTGAG TAAACACCCA TTCCACTCCA CGACCGCAAG CT - |
| #CCATCTTG 1500 |
| - - CGCGTGGCGT CTCTGCGATG AACATCCGAA ACTGACGTTC TGTTACAGAC TG - |
| #GTCACTTG 1560 |
| - - ATCTACCAGT GCGGTGGTAT CGACAAGCGT ACCATTGAGA AGTTCGAGAA GG - |
| #TAAGCTTC 1620 |
| - - GTTCCTTAAA TCTCCAGACG CGAGCCCAAT CTTTGCCCAT CTGCCCAGCA TC - |
| #TGGCGAAC 1680 |
| - - GAATGCTGTG CCGACACGAT TTTTTTTTTC ATCACCCCGC TTTCTCCTAC CC - |
| #CTCCTTCG 1740 |
| - - AGCGACGCAA ATTTTTTTTG CTGCCTTACG AGTTTTAGTG GGGTCGCACC TC - |
| #ACAACCCC 1800 |
| - - ACTACTGCTC TCTGGCCGCT CCCCAGTCAC CCAACGTCAT CAACGCAGCA GT - |
| #TTTCAATC 1860 |
| - - AGCGATGCTA ACCATATTCC CTCGAACAGG AAGCCGCCGA ACTCGGCAAG GG - |
| #TTCCTTCA 1920 |
| - - AGTACGCGTG GGTTCTTGAC AAGCTCAAGG CCGAGCGTGA GCGTGGTATC AC - |
| #CATCGACA 1980 |
| - - TTGCCCTCTG GAAGTTCGAG ACTCCCAAGT ACTATGTCAC CGTCATTGGT AT - |
| #GTTGGCAG 2040 |
| - - CCATCACCTC ACTGCGTCGT TGACACATCA AACTAACAAT GCCCTCACAG AC - |
| #GCTCCCGG 2100 |
| - - CCACCGTGAC TTCATCAAGA ACATGATCAC TGGTACTTCC CAGGCCGACT GC - |
| #GCTATCCT 2160 |
| - - CATCATCGCT GCCGGTACTG GTGAGTTCGA GGCTGGTATC TCCAAGGATG GC - |
| #CAGACCCG 2220 |
| - - TGAGCACGCT CTGCTCGCCT ACACCCTGGG TGTCAAGCAG CTCATCGTCG CC - |
| #ATCAACAA 2280 |
| - - GATGGACACT GCCAACTGGG CCGAGGCTCG TTACCAGGAA ATCATCAAGG AG - |
| #ACTTCCAA 2340 |
| - - CTTCATCAAG AAGGTCGGCT TCAACCCCAA GGCCGTTGCT TTCGTCCCCA TC - |
| #TCCGGCTT 2400 |
| - - CAACGGTGAC AACATGCTCA CCCCCTCCAC CAACTGCCCC TGGTACAAGG GC - |
| #TGGGAGAA 2460 |
| - - GGAGACCAAG GCTGGCAAGT TCACCGGCAA GACCCTCCTT GAGGCCATCG AC - |
| #TCCATCGA 2520 |
| - - GCCCCCCAAG CGTCCCACGG ACAAGCCCCT GCGTCTTCCC CTCCAGGACG TC - |
| #TACAAGAT 2580 |
| - - CGGTGGTATC GGAACAGTTC CCGTCGGCCG TATCGAGACT GGTGTCCTCA AG - |
| #CCCGGTAT 2640 |
| - - GGTCGTTACC TTCGCTCCCT CCAACGTCAC CACTGAAGTC AAGTCCGTCG AG - |
| #ATGCACCA 2700 |
| - - CGAGCAGCTC GCTGAGGGCC AGCCTGGTGA CAACGTTGGT TTCAACGTGA AG - |
| #AACGTTTC 2760 |
| - - CGTCAAGGAA ATCCGCCGTG GCAACGTTGC CGGTGACTCC AAGAACGACC CC - |
| #CCCATGGG 2820 |
| - - CGCCGCTTCT TTCACCGCCC AGGTCATCGT CATGAACCAC CCCGGCCAGG TC - |
| #GGTGCCGG 2880 |
| - - CTACGCCCCC GTCCTCGACT GCCACACTGC CCACATTGCC TGCAAGTTCG CC - |
| #GAGCTCCT 2940 |
| - - CGAGAAGATC GACCGCCGTA CCGGTAAGGC TACCGAGTCT GCCCCCAAGT TC - |
| #ATCAAGTC 3000 |
| - - TGGTGACTCC GCCATCGTCA AGATGATCCC CTCCAAGCCC ATGTGCGTTG AG - |
| #GCTTTCAC 3060 |
| - - CGACTACCCT CCCCTGGGTC GTTTCGCCGT CCGTGACATG CGCCAGACCG TC - |
| #GCTGTCGG 3120 |
| - - TGTCATCAAG GCCGTCGAGA AGTCCTCTGC CGCCGCCGCN AAGGTCACCA AG - |
| #TCCGCTGC 3180 |
| - - CAAGGCCGCC AAGAAATAAG CGATACCCAT CATCAACACC TGATGTTCTG GG - |
| #GTCCCTCG 3240 |
| - - TGAGGTTTCT CCAGGTGGGC ACCACCATGC GCTCACTTCT ACGACGAAAC GA - |
| #TCAATGTT 3300 |
| - - GCTATGCATG AGSACTCGAC TATGAATCGA GGCACGGTTA ATTGAGAGGC TG - |
| #GGAATAAG 3360 |
| - - GGTTCCATCA GAACTTCTCT GGGAATGCAA AACAAAAGGG AACAAAAAAA CT - |
| #AGATAGAA 3420 |
| - - GTGAATTCAT GACTTCGACA ACCAAAAAAA AAAAAAAAAA A - # |
| - # 3461 |
| - - - - (2) INFORMATION FOR SEQ ID NO:2: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 2336 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| - - (ix) FEATURE: |
| (A) NAME/KEY: misc-- - #feature |
| (B) LOCATION: 1..700 |
| (D) OTHER INFORMATION: - #/note= "The first 700 bases of the |
| nucleotide - #sequence are vector pSP73 sequences and |
| are not - #part of the promoter sequence." |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: |
| - - GCCAGTGGCG ATAAGTCGTG TCTTCCGGGT TGGACTCAAG ACGATAGTTA CC - |
| #GGATAAGG 60 |
| - - CGCAGCGGTC GGGCTGAACG GGGGGTTCGT GCACACAGCC CAGCTTGGAG CG - |
| #AACGACCT 120 |
| - - ACACCGAACT GAGATACCTA CAGCGTGAGC TATGAGAAAG CGCCACGCTT CC - |
| #CGAAGGGA 180 |
| - - GAAAGGCGGA CAGGTATCCG GTAAGCGGCA GGGTCGGAAC AGGAGAGAGA AA - |
| #GAGGGANN 240 |
| - - TTCCAGGGGG AAACGCCTGG TATCTTTATA GTCCTGTTGG GTTTCGCCAC CT - |
| #CTGACTTG 300 |
| - - AGCGTCGATT TTTGTGATGC TCGTCAGGGG GNGGAGCCTA TGGAAAAACG CC - |
| #AGCAACGC 360 |
| - - GGCCTTTTTA CGGTTCCTGG CCTTTTGCTG GCCTTTTGCT CACATGTTCT TT - |
| #CCTGCGTT 420 |
| - - ATCCCCTGAT TCTGTGGATA ACCGTATTAC CGCCTTTGAG TGAGCTGATA CC - |
| #GCTCGCCG 480 |
| - - CAGCCGAACG ACCGAGCGCA GCGAGTCAGT GAGCGAGGAA GCGGAAGAGC GC - |
| #CCAATACG 540 |
| - - CAAACCGCCT CTCCCCGCGC GTTGGCCGAT TCATTAATGC AGGTTAACCT GG - |
| #CTTATCGA 600 |
| - - AATTAATACG ACTCACTATA GGGAGACCGG CCTCGAGCAG CTGAAGCTTG CA - |
| #TGCCTGCA 660 |
| - - GGTCGACTCT AGAGGATCCC CGGGTACCGA GCTCGAATTC GGTCTGAAGG AC - |
| #GTGGAATG 720 |
| - - ATGGACTTAA TGACAAGAGT TGCCTGGCTA TTGAGCTCTG GTACATGGAT CT - |
| #CGAACTGA 780 |
| - - GAGCGTACAA GTTACATGTA GTAAATCTAG TAGATCTCGC TGAAAGCCCT CT - |
| #TTCCCGGT 840 |
| - - AGAAACACCA CCAGCGTCCC GTAGGACAAG ATCCTGTCGA TCTGAGCACA TG - |
| #AATTGCTT 900 |
| - - CCCTGGATCT GGCGCTGCAT CTGTTTCCCC AGACAATGAT GGTAGCAGCG CA - |
| #TGGAAGAA 960 |
| - - CCCGGTTGTT CGGAATGTCC TTGTGCTAAC AGTGGCATGA TTTTACGTTG CG - |
| #GCTCATCT 1020 |
| - - CGCCTTGGCA CCGGACCTCA GCAAATCTTG TCACAACAGC AATCTCAAAC AG - |
| #CCTCATGG 1080 |
| - - TTCCCAGATT CCCTGATTCA GAACTCTAGA GCGGCAGATG TCAAACGATT CT - |
| #GACCTAGT 1140 |
| - - ACCTTGAGCA TCCCTTTCGG ATCCGGCCCA TGTTCTGCCT GCCCTTCTGA GC - |
| #ACAGCAAA 1200 |
| - - CAGCCCAAAA GGCGCCGGCC GATTCCTTTC CCGGGATGCT CCGGAGTGGC AC - |
| #CACCTCCC 1260 |
| - - AAAACAAGCA ACCTTGAACC CCCCCCCCAA ATCAACTGAA GCGCTCTTCG CC - |
| #TAACCAGC 1320 |
| - - ATAAGCCCCC CCCAGGATCG TTAGGCCAAG TGGTAGGGCC AGCCAATTAG CG - |
| #AGNGGCCA 1380 |
| - - TTTGGAGGTC ATGGGCGCAG AATGTCCTGA CAGTGGTATG ATATTGACTG CC - |
| #CGGTGTGT 1440 |
| - - GTGGCATCTG GCCATAATCG CAGGCTGAGG CGAGGAAGTC TCGTGAGGAT GT - |
| #CCCGACTT 1500 |
| - - TGACATCATG AGGGAGTGAG AAACTGAAGA GAAGGAAAGC TTCGAAGGTT CG - |
| #ATAAGGGA 1560 |
| - - TGATTTGCAT GGCGGGCGAC AGGATGCGAT GGCTCGTTGG GATACATAAT GC - |
| #TTGGGTTG 1620 |
| - - GAAGCGATTC CAGGTCGTCT TTTTTTGGTT CATCATCACA GCATCAACAA GC - |
| #AACGATAC 1680 |
| - - AAGCAATCCA CTGAGGATTA CCTCTCAACT CAACCACTTT CCAAACCATC TC - |
| #AACTCCCT 1740 |
| - - AAGATTCTTT CAGTGTATTA TCACTAGGAT TTTTCCCAAG CCGGCTTCAA AA - |
| #CACACAGA 1800 |
| - - TAAACCACCA ACTCTACAAC CAAAGACTTT TTGATCAATC CAACAACTTC TC - |
| #TCAACATG 1860 |
| - - TCTGCTGCAA CCGTCACCCG CACTGCAACC GCCGCTGTTC GCAGACCCGG CT - |
| #TCTTCATG 1920 |
| - - CAAGTCCGAC GGATGGGACG CTCATTCGAG CACCAGCCCT TTGAGCGACT CT - |
| #CCGCCACC 1980 |
| - - ATGAAGCCTG CACGACCCGA CTATGCTAAG CAAGTCGTCT GGACGGCTGG CA - |
| #AGTTTGTC 2040 |
| - - ACTTATGTTC CTCTTTTCGG CGCCATGCTT ACCTGGCCTG CGCTCGCCAA ST - |
| #GGGCTCTG 2100 |
| - - GACGGACACA TCGGACGGTG GTAAAAGATC AGACTCTTGT CGAGGCAACG GG - |
| #GAATAGAC 2160 |
| - - AGGACAGCAA AAAAGATATC TCCGGATAGA AGTGTCCATC TTTCGACTTG TA - |
| #TATATATA 2220 |
| - - TATGCTATAC TCTGGGGGCG TTTGGATGGA CTTTGGGCAC GAAGCATACT TT - |
| #GGCGCAAC 2280 |
| - - GCAGATACTT TAATCTGATT CCTTTTGTTA ATTCAAAAAA AAAAAAAAAA AA - #AAAA |
| 2336 |
| - - - - (2) INFORMATION FOR SEQ ID NO:3: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 2868 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: |
| - - TTTGTATGGC TGGATCTCGA AAGGCCCTTG TCATCGCCAA GCGTGGCTAA TA - |
| #TCGAATGA 60 |
| - - GGGACACCCA CTTGCATATC TCCTGATCAT TCAAACGACA AGTGTGAGGT AG - |
| #GCAATCCT 120 |
| - - CGTATCCCAT TGCTGGGCTG AAAGCTTCAC ACGTATCGCA TAAGCGTCTC CA - |
| #ACCAGTGC 180 |
| - - TTAGGTGACC CTTAAGGATA CTTACAGTAA GACTGTATTA AGTCAGTCAC TC - |
| #TTTCACTC 240 |
| - - GGGCTTTGAA TACGATCCTC AATACTCCCG ATAACAGTAA GAGGATGATA CA - |
| #GCCTGCAG 300 |
| - - TTGGCAAATG TAAGCGTAAT TAAACTCAGC TGAACGGCCC TTGTTGAAAG TC - |
| #TCTCTCGA 360 |
| - - TCAAAGCAAA GCTATCCACA GACAAGGGTT AAGCAGGCTC ACTCTTCCTA CG - |
| #CCTTGGAT 420 |
| - - ATGCAGCTTG GCCAGCATCG CGCATGGCCA ATGATGCACC CTTCACGGCC CA - |
| #ACGGATCT 480 |
| - - CCCGTTAAAC TCCCCTGTAA CTTGGCATCA CTCATCTGTG ATCCCAACAG AC - |
| #TGAGTTGG 540 |
| - - GGGCTGCGGC TGGCGGATGT CGGAGCAAAG GATCACTTCA AGAGCCCAGA TC - |
| #CGGTTGGT 600 |
| - - CCATTGCCAA TGGATCTAGA TTCGGCACCT TGATCTCGAT CACTGAGACA TG - |
| #GTGAGTTG 660 |
| - - CCCGGACGCA CCACAACTCC CCCTGTGTCA TTGAGTCCCC ATATGCGTCT TC - |
| #TCAGCGTG 720 |
| - - CAACTCTGAG ACGGATTAGT CCTCACGATG AAATTAACTT CCAGCTTAAG TT - |
| #CGTAGCCT 780 |
| - - TGAATGAGTG AAGAAATTTC AAAAACAAAC TGAGTAGAGG TCTTGAGCAG CT - |
| #GGGGTGGT 840 |
| - - ACGCCCCTCC TCGACTCTTG GGACATCGTA CGGCAGAGAA TCAACGGATT CA - |
| #CACCTTTG 900 |
| - - GGTCGAGATG AGCTGATCTC GACAGATACG TGCTTCACCA CAGCTGCAGC TA - |
| #CCTTTGCC 960 |
| - - CAACCATTGC GTTCCAGGAT CTTGATCTAC ATCACCGCAG CACCCGAGCC AG - |
| #GACGGAGA 1020 |
| - - GAACAATCCG GCCACAGAGC AGCACCGCCT TCCAACTCTG CTCCTGGCAA CG - |
| #TCACACAA 1080 |
| - - CCTGATATTA GATATCCACC TGGGTGATTG CCATTGCAGA GAGGTGGCAG TT - |
| #GGTGATAC 1140 |
| - - CGACTGGCCA TGCAAGACGC GGCCGGGCTA GCTGAAATGT CCCCGAGAGG AC - |
| #AATTGGGA 1200 |
| - - GCGTCTATGA CGGCGTGGAG ACGACGGGAA AGGACTCAGC CGTCATGTTG TG - |
| #TTGCCAAT 1260 |
| - - TTGAGATTGT TGACCGGGAA AGGGGGGACG AAGAGGATGG CTGGGTGAGG TG - |
| #GTATTGGG 1320 |
| - - AGGATGCATC ATTCGACTCA GTGAGCGATG TAGAGCTCCA AGAATATAAA TA - |
| #TCCCTTCT 1380 |
| - - CTGTCTTCTC AAAATCTCCT TCCATCTTGT CCTTCATCAG CACCAGAGCC AG - |
| #CCTGAACA 1440 |
| - - CCTCCAGTCA ACTTCCCTTA CCAGTACATC TGAATCAACA TCCATTCTTT GA - |
| #AATCTCAC 1500 |
| - - CACAACCACC ATCTTCTTCA AAATGAAGTT CTTCGCCATC GCCGCTCTCT TT - |
| #GCCGCCGC 1560 |
| - - TGCCGTTGCC CAGCCTCTCG AGGACCGCAG CAACGGCAAC GGCAATGTTT GC - |
| #CCTCCCGG 1620 |
| - - CCTCTTCAGC AACCCCCAGT GCTGTGCCAC CCAAGTCCTT GGCCTCATCG GC - |
| #CTTGACTG 1680 |
| - - CAAAGTCCGT AAGTTGAGCC ATAACATAAG AATCCTCTTG ACGGAAATAT GC - |
| #CTTCTCAC 1740 |
| - - TCCTTTACCC CTGAACAGCC TCCCAGAACG TTTACGACGG CACCGACTTC CG - |
| #CAACGTCT 1800 |
| - - GCGCCAAAAC CGGCGCCCAG CCTCTCTGCT GCGTGGCCCC CGTTGTAAGT TG - |
| #ATGCCCCA 1860 |
| - - GCTCAAGCTC CAGTCTTTGG CAAACCCATT CTGACACCCA GACTGCAGGC CG - |
| #GCCAGGCT 1920 |
| - - CTTCTGTGCC AGACCGCCGT CGGTGCTTGA GATGCCCGCC CGGGGTCAAG GT - |
| #GTGCCCGT 1980 |
| - - GAGAAAGCCC ACAAAGTGTT GATGAGGACC ATTTCCGGTA CTGGGAAAGT TG - |
| #GCTCCACG 2040 |
| - - TGTTTGGGCA GGTTTGGGCA AGTTGTGTAG ATATTCCATT CGTACGCCAT TC - |
| #TTATTCTC 2100 |
| - - CAATATTTCA GTACACTTTT CTTCATAAAT CAAAAAGACT GCTATTCTCT TT - |
| #GTGACATG 2160 |
| - - CCGGAAGGGA ACAATTGCTC TTGGTCTCTG TTATTTGCAA GTAGGAGTGG GA - |
| #GATTCGCC 2220 |
| - - TTAGAGAAAG TAGAGAAGCT GTGCTTGACC GTGGTGTGAC TCGACGAGGA TG - |
| #GACTGAGA 2280 |
| - - GTGTTAGGAT TAGGTCGAAC GTTGAAGTGT ATACAGGATC GTCTGGCAAC CC - |
| #ACGGATCC 2340 |
| - - TATGACTTGA TGCAATGGTG AAGATGAATG ACAGTGTAAG AGGAAAAGGA AA - |
| #TGTCCGCC 2400 |
| - - TTCAGCTGAT ATCCACGCCA ATGATACAGC GATATACCTC CAATATCTGT GG - |
| #GAACGAGA 2460 |
| - - CATGACATAT TTGTGGGAAC AACTTCAAAC AGCGAGCCAA GACCTCAATA TG - |
| #CACATCCA 2520 |
| - - AAGCCAAACA TTGGCAAGAC GAGAGACAGT CACATTGTCG TCGAAAGATG GC - |
| #ATCGTACC 2580 |
| - - CAAATCATCA GCTCTCATTA TCGCCTAAAC CACAGATTGT TTGCCGTCCC CC - |
| #AACTCCAA 2640 |
| - - AACGTTACTA CAAAAGACAT GGGCGAATGC AAAGACCTGA AAGCAAACCC TT - |
| #TTTGCGAC 2700 |
| - - TCAATTCCCT CCTTTGTCCT CGGAATGATG ATCCTTCACC AAGTAAAAGA AA - |
| #AAGAAGAT 2760 |
| - - TGAGATAATA CATGAAAAGC ACAACGGAAA CGAAAGAACC AGGAAAAGAA TA - |
| #AATCTATC 2820 |
| - - ACGCACCTTG TCCCCACACT AAAAGCAACA GGGGGGGTAA AATGAAAT - # |
| 2868 |
| - - - - (2) INFORMATION FOR SEQ ID NO:4: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 2175 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: |
| - - AAAAAGCTAG AACGAGACGA TTCCGGCCCG GCAAACCAGG CCGAGTGACG GG - |
| #AGCATTTC 60 |
| - - CATGATTTCA CTCGGCAAAC TCTGGCTACA ATTTTCAGGC GGCGAGTTCC GA - |
| #TACAAGGG 120 |
| - - AAATCTATTA CCCACAGACG AACGGGAATC GGTGATGAGT GGTTTCTTGT AA - |
| #GTCAACAT 180 |
| - - TGAGCTAGAT AATTCCGGGC GAGATCAAGA TGCCATACTT TGATTGATGA AA - |
| #AATCAATG 240 |
| - - TCAGGCGTAA GTCTCTTCAA GCTCGCCCAG TCCTCTGTAT GTAACAGCAA TC - |
| #GCAATTCC 300 |
| - - GAAATGTGCC GAGCCAATGG AACATGCGTG TCTTTCTCTT TTCACACACA TC - |
| #CAGTTCGA 360 |
| - - GAGTCTTCTC TTCATCGTTT CATCGAATCC CTTCCCCTCC AGCTATTCAC CC - |
| #AGCCGAGC 420 |
| - - CCTTCAGCGC ACCAGCGTAT GTATGTACCC TCGGCTAAGA CGCAACAGAA GC - |
| #ATCATCAA 480 |
| - - TATACCTGAT GTACTACTAT CTACTATGAA GCCCAAAAAC CCCTTCGCAG CC - |
| #CAAATGTA 540 |
| - - ACCCAAGCAA CGAATCCCCA ATAAGAGACA ATCCTCAGTG ACCCCCAGAA GA - |
| #GCACAGAA 600 |
| - - TCGAGCTGGT CCTGGTGGGT CGCATTGAGA CCGGTGGAGA TGCGTTCGAT TC - |
| #GACTGCCG 660 |
| - - GAGCTCCCGG GAAGCCGGCA GATGGTCCCA TGCGATGCCC TGCACCGTTT TT - |
| #GTGAATCG 720 |
| - - TCGGCATCGC GAGAAGTGGC CTGCTATGAC GTCGCTTGCA GCTTGGCCGC TC - |
| #TGTTCGAA 780 |
| - - GTTTTTCGAT GTTTTTCTTC ATGCGGGAGA AAGAAAACAT CAGATGACAT GA - |
| #TTATCCGA 840 |
| - - ATGGATGGCG GGAGTTATCG TGGTGACGGC TGCTTCATGA GATGAGTATA AA - |
| #TGAGCTTG 900 |
| - - TTCGCTCAGC GTGTCATGGA TCTTGTCCAG CTCCAAAGCA TCGGCTTCAG CA - |
| #TCCATCCG 960 |
| - - CTTGAACAGA CAGGCACCAG CTTGAATCAG AAGCATACCC TTGATTTGAT AC - |
| #TCTCTTGG 1020 |
| - - GAAAAAACAC CACCATCTGT GTAATACTTT GATACCCCCA AAGCTCAAAC GA - |
| #CCGCTTGT 1080 |
| - - ACATACAATA ACACCGCCAC AATGTTCGCC AACTTGACGC ACGCTACCCT GC - |
| #GATTCATC 1140 |
| - - GCCTTCTTCA ACCACCTGAT GATCCTGGCC TCATCAGCCA TCGTCACCGG CC - |
| #TCGTATCC 1200 |
| - - TGGTTCCTCG ACAAGTACGA CTACCGCGGC GTGAACATTG TCTACCAGGA AG - |
| #TCATCGTA 1260 |
| - - TGTCCTCCCA AGCACCACAT CAAACACACC CCATACCTTG GCTCTCCTCA GC - |
| #TCCGTCGA 1320 |
| - - AGCACATAAT ACTAACGCAT GCAACAACTA GGCCACCATA ACTCTGGGCT TC - |
| #TGGCTCGT 1380 |
| - - TGGTGCCGTC TTGCCCCTCG TTGGCAGATA CCGCGGCCAC CTGGCCCCTC TC - |
| #AACCTCAT 1440 |
| - - CTTCTCCTAC CTCTGGCTCA CCTCTTTCAT CTTCTCCGCG CAGGACTGGA GC - |
| #AGCGACAA 1500 |
| - - GTGCAGCTTC GGCCAGCCTG GCGAGGGCCA CTGCAGCCGC AAGAAGGCCA TT - |
| #GAATCCTT 1560 |
| - - CAACTTTATC GCATTGTAAG TGCCTACAAG TAATTTGCTA TGTATATGGG AG - |
| #AGAGAGAG 1620 |
| - - AAGAAGAAGA ATATGGCTCT AACATGGCAT CTCTACAGCT TCTTCCTCCT CT - |
| #GCAACACC 1680 |
| - - CTGGTTGAGA TGCTCCTGCT CCGCGCCGAG TATGCTACCC CCGTTGCTGC TG - |
| #CTCACAAC 1740 |
| - - AAGGAGATTT CTGCCGGCCG CCCCTCTGAC AACTCTGTCT AAATAACAAT AG - |
| #ACATGCAT 1800 |
| - - AGATGAACGG AGACCACTTC TACTTTCTTT GCGAGTTCCT GATCCGTTGA CC - |
| #TGCAGGTC 1860 |
| - - GACBBBBBCC GCGCTCGCAT GGTTCATCTG CTACAACAAC ACAATGACAA TC - |
| #CGAACCAG 1920 |
| - - TCAATAAACC TCGACAACAC GACGAGTACT TTTGCGGATA GAAAGATACC CA - |
| #TTACACAG 1980 |
| - - GAGATCAAAT GGGGAAATTG GAAGTGTATG GATGGACGCC CGTGTATAAT GA - |
| #GGTTGTGA 2040 |
| - - ACGGGATGGG AGGCAATGAA TAATGGATAA TGAGGTAATG GATAGATTCG GT - |
| #CGTTTTGA 2100 |
| - - TACCACAGCT GCACTCTGCT CTACGTCTGT CATTAATGAT ACATACAAAT GA - |
| #TACCTTAT 2160 |
| - - ACGCTAAAAA AAAAA - # - # |
| - # 2175 |
| - - - - (2) INFORMATION FOR SEQ ID NO:5: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 2737 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: |
| - - TCTAGAATCT CTTCGAGATG GCCGAGAAAG GCTTGTTTTT CTCTCCTTCT TC - |
| #AAACTGGC 60 |
| - - CACTGTTTGT TTTCAAACTT GGGGTTTCGT GGGGCTTTTG GGGGCATGTC TG - |
| #CCAGGTCT 120 |
| - - CCCGTAGGCT GGACAGCCAA AGCCTCACTA CAAACAGGCA GTTGTCAATA GA - |
| #TTGATGTC 180 |
| - - TGAGATGGAT GGTTTTATGT TTGGGGGAGG TCATGTATGT ATTTATCTAT AT - |
| #TTGCAAAG 240 |
| - - ATGATCCATG AGTCAGACTT GCACAGGTTT CTCGTGCGCT GGATAAATCT TG - |
| #TTGGAGTG 300 |
| - - CGGGTGAGGT GGTGGATGGC ATTCAACCCA CAGCAACACT TGCCCAGGGG GA - |
| #TGTACTGC 360 |
| - - AGCGATTTGT TTCCCTTCGA GTATTAGATG ATGATGCCGA ACAGACAAAT TT - |
| #GAGCCTCG 420 |
| - - CTGCTCTCGG ATGTCGGGTT TCTCTTGTGT GCCGGTGATG TGTGATGGCC TG - |
| #GCCCGCAA 480 |
| - - AGAGAGCGAA AAACATGCTC AAAATGTAGC ACACGGCGAC TTCTCGGACA CT - |
| #TGCGTACC 540 |
| - - TTGAGAGACA AGCAGACTAC AGGGATGACG AGTAATACGA CAGAGCGATA CG - |
| #ACACAGCT 600 |
| - - ATACGACACA GCTAAGAAAA TAAAGGTATT AGTACTACTA ATTGATTACC TA - |
| #CTACCTAG 660 |
| - - ATATATACTA TACCTTATAT TTTATATGTG TGTGTGTGTG TATGTATATG CC - |
| #TTACCTTA 720 |
| - - TGCTTCGCAA AGAAGAGAAA CTAAAACGCC TCCTGGCTAC CTACCTACCT CT - |
| #ACCTTGTA 780 |
| - - AGAGATGGAA TAATGTGGCC GCGCGTAAAG TAGGTACTGG ATATACAGGT CC - |
| #TGAACATG 840 |
| - - GCCCTGAATC CTGCCAGGCA GCCACCTCAC CCCTTCCGCA GGTATTTATG TA - |
| #GCCCACAG 900 |
| - - CTCCTCCAGA GACGATGCCG AGATGCCTCA TGCAGTCTAC CTACAAAGCC AG - |
| #CAGTTTCA 960 |
| - - CGCTTGACTC TCACTCTTGA TTGAATTCCC TCCCTCCCAT AATACCAATT GG - |
| #CGTTCAAC 1020 |
| - - GATTGCCAGC AGAATGGCCG CCCAACACGA CGTCGAGGCC ATGGCAAAGT CC - |
| #ATGTCCGA 1080 |
| - - CTTTTTCAAG GACACGGCCC AAAAGCAGGA CTCGACCAAG CATGACTTTG TC - |
| #CAAGCCTC 1140 |
| - - GCACGGCATC ATGAGGGCCA TTGTCGAGCC GCTCGTCACC CAGATGGGCT TC - |
| #CGCGAGAC 1200 |
| - - CCTCACCGAG CCCGTCGTCT TGCTCGACAG CGCGTGCGGA GCGGGCGTGC TG - |
| #ACGCAGGA 1260 |
| - - GGTGCAGGCG GCGCTGCCAA AGGAGCTTCT GGAGAGGAGC TCGTTTACGT GT - |
| #GCGGACAA 1320 |
| - - TGCCGAGGGC TTGGTGGACG TGGTGAAGAG GAGGATTGAT GAGGAGAAGT GG - |
| #GTGAATGC 1380 |
| - - AGAGGCCAAG GTCCTTGATG CCCTGGTGAG TATATACATA TATATCTATA TC - |
| #TATATAGA 1440 |
| - - TATATATATG CCTTTGACTC CCCCCTTTAC ATGTCCTACG GCTGCTGATT GA - |
| #TTGATTGA 1500 |
| - - TGTGGTGATG GTGATGTCCC AGAACACGGG GCTCCCAGAC AACTCCTTCA CC - |
| #CATGTGGG 1560 |
| - - CATTGCCCTG GCACTGCACA TCATCCCCGA TCCAGATGCC GTCGTCAAAG GT - |
| #AAACAATC 1620 |
| - - ACCAGCGTCA CTGCAAAGAG AGATTACGGG ATATCATATA CTGAAACCAA AG - |
| #CCCAGACT 1680 |
| - - GCATCAGAAT GCTCAAGCCA GGCGGCATCT TTGGCGCATC GACATGGCCC AA - |
| #GGCCAGCG 1740 |
| - - CCGACATGTT CTGGATCGCC GACATGCGCA CCGCCCTGCA GTCGCTCCCC TT - |
| #TGACGCGC 1800 |
| - - CGCTGCCAGA CCCGTTCCCC ATGCAGCTGC ACACCTCGGG CCACTGGGAC GA - |
| #CGCCGCCT 1860 |
| - - GGGTCGAGAA GCATCTCGTC GAGGATCTGG GGCTGGCCAA CGTCTGTGTG AG - |
| #GGAGCCGG 1920 |
| - - CGGGCGAGTA CAGCTTTGCG AGCGCGGACG AGTTCATGGC GACGTTTCAG AT - |
| #GATGCTGC 1980 |
| - - CGTGGATTAT GAAGACGTTT TGGAGCGAGG AGGTGAGGGA GAAGCATTCG GT - |
| #CGACGAGG 2040 |
| - - TCAAGGAGTT GGTGAAGAGG CATCTGGAGG ACAAGTATGG GGGGAAGGGA TG - |
| #GACCATTA 2100 |
| - - AGTGGCGGGT GATTACCATG ACTGCGACTG CGAGCAAGTG AGGGAGGGCA TC - |
| #TGCTCATG 2160 |
| - - ATTATGTGAC AGCGAGCCAG TAGAGAGCCA TATTGTTGTC TTCAGAATGT GA - |
| #GGACCGTG 2220 |
| - - ATGGTTGGTG TTTGTTGGAG TGATAACTCG TGGGTGTTGC TATTTGCATG TG - |
| #AGACGATG 2280 |
| - - AACCATGCGC ACCAGCCACA ATCACTGTCC CCCACCTTAC CTACCAACTT CA - |
| #AGTTACCA 2340 |
| - - CCTTACCTTT ACCTGATCTA GCACTGTGGC GCAGCTTGGT TTGACTGCTA GG - |
| #TACCTACC 2400 |
| - - TAGTAGTAAT CAGGTACATT CTTCATCCCT GTGTCCTGGT GTCGCAGTTG CA - |
| #GCTTGTCT 2460 |
| - - TATCGCTGTG GCCACGCATC GAGTGGCAGC ATCTTCAACT TCAAGTCCCG TC - |
| #GGTCGCAC 2520 |
| - - TCTGGCCACG TCGCAGATGG ATCGCAGCGG GATCTGAACC GCTCGCTCGG CA - |
| #ACTGATAC 2580 |
| - - CAAGTCAACA AACACACGAG ACGACGGGAC GCTGATATAA NNNNGAGGAG GG - |
| #TAAGAGAA 2640 |
| - - CTCTACGAGG GGCGGAAACT TGGTCCGACA ATTTCCCTCC CATCTTCACC CT - |
| #CGACTCGA 2700 |
| - - ACTCGAACTC GATAGCCGCA CCCTCGACCG ATTGCCC - # |
| - # 2737 |
| - - - - (2) INFORMATION FOR SEQ ID NO:6: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 43 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: |
| - - ACCGGAATTC ATATCTAGAG GAGCCCGCGA GTTTGGATAC GCC - # |
| - # 43 |
| - - - - (2) INFORMATION FOR SEQ ID NO:7: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 34 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: |
| - - ACCGCCGCGG TTTGACGGTT TGTGTGATGT AGCG - # - |
| # 34 |
| - - - - (2) INFORMATION FOR SEQ ID NO:8: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 41 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: |
| - - TCTTCAAGAA TTGCTCGACC AATTCTCACG GTGAATGTAG G - # |
| - # 41 |
| - - - - (2) INFORMATION FOR SEQ ID NO:9: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 73 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: |
| - - ACACATCTAG AGGTGACCTA GGCATTCTGG CCACTAGATA TATATTTAGA AG - |
| #GTTCTTGT 60 |
| - - AGCTCAAAAG AGC - # - # |
| - # 73 |
| - - - - (2) INFORMATION FOR SEQ ID NO:10: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 38 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: |
| - - GGGAATTCTC TAGAAACGCG TTGGCAAATT ACGGTACG - # |
| - # 38 |
| - - - - (2) INFORMATION FOR SEQ ID NO:11: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 43 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: |
| - - GGGAATTCGG TCACCTCTAA ATGTGTAATT TGCCTGCTTG ACC - # |
| - # 43 |
| - - - - (2) INFORMATION FOR SEQ ID NO:12: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 73 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: |
| - - GGGAATTCGG TCACCTCTAA ATGTGTAATT TGCCTGCTTG ACCGATCTAA AC - |
| #TGTTCGAA 60 |
| - - GCCCGAATGT AGG - # - # |
| - # 73 |
| - - - - (2) INFORMATION FOR SEQ ID NO:13: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 45 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: |
| - - GGGAATTCTT CTAGATTGCA GAAGCACGGC AAAGCCCACT TACCC - # |
| - #45 |
| - - - - (2) INFORMATION FOR SEQ ID NO:14: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 47 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: |
| - - TAGCGAATTC TAGGTCACCT CTAAAGGTAC CCTGCAGCTC GAGCTAG - # |
| 47 |
| - - - - (2) INFORMATION FOR SEQ ID NO:15: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 26 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: |
| - - GGGAATTCAT GATGCGCAGT CCGCGG - # - # |
| 26 |
| - - - - (2) INFORMATION FOR SEQ ID NO:16: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 1588 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: |
| - - CCCCCCTATC TTAGTCCTTC TTGTTGTCCC AAAATGGCGC CCTCAGTTAC AC - |
| #TGCCGTTG 60 |
| - - ACCACGGCCA TCCTGGCCAT TGCCCGGCTC GTCGCCGCCC AGCAACCGGG TA - |
| #CCAGCACC 120 |
| - - CCCGAGGTCC ATCCCAAGTT GACAACCTAC AAGTGTACAA AGTCCGGGGG GT - |
| #GCGTGGCC 180 |
| - - CAGGACACCT CGGTGGTCCT TGACTGGAAC TACCGCTGGA TGCACGACGC AA - |
| #ACTACAAC 240 |
| - - TCGTGCACCG TCAACGGCGG CGTCAACACC ACGCTCTGCC CTGACGAGGC GA - |
| #CCTGTGGC 300 |
| - - AAGAACTGCT TCATCGAGGG CGTCGACTAC GCCGCCTCGG GCGTCACGAC CT - |
| #CGGGCAGC 360 |
| - - AGCCTCACCA TGAACCAGTA CATGCCCAGC AGCTCTGGCG GCTACAGCAG CG - |
| #TCTCTCCT 420 |
| - - CGGCTGTATC TCCTGGACTC TGACGGTGAG TACGTGATGC TGAAGCTCAA CG - |
| #GCCAGGAG 480 |
| - - CTGAGCTTCG ACGTCGACCT CTCTGCTCTG CCGTGTGGAG AGAACGGCTC GC - |
| #TCTACCTG 540 |
| - - TCTCAGATGG ACGAGAACGG GGGCGCCAAC CAGTATAACA CGGCCGGTGC CA - |
| #ACTACGGG 600 |
| - - AGCGGCTACT GCGATGCTCA GTGCCCCGTC CAGACATGGA GGAACGGCAC CC - |
| #TCAACACT 660 |
| - - AGCCACCAGG GCTTCTGCTG CAACGAGATG GATATCCTGG AGGGCAACTC GA - |
| #GGGCGAAT 720 |
| - - GCCTTGACCC CTCACTCTTG CACGGCCACG GCCTGCGACT CTGCCGGTTG CG - |
| #GCTTCAAC 780 |
| - - CCCTATGGCA GCGGCTACAA AAGCTACTAC GGCCCCGGAG ATACCGTTGA CA - |
| #CCTCCAAG 840 |
| - - ACCTTCACCA TCATCACCCA GTTCAACACG GACAACGGCT CGCCCTCGGG CA - |
| #ACCTTGTG 900 |
| - - AGCATCACCC GCAAGTACCA GCAAAACGGC GTCGACATCC CCAGCGCCCA GC - |
| #CCGGCGGC 960 |
| - - GACACCATCT CGTCCTGCCC GTCCGCCTCA GCCTACGGCG GCCTCGCCAC CA - |
| #TGGGCAAG 1020 |
| - - GCCCTGAGCA GCGGCATGGT GCTCGTGTTC AGCATTTGGA ACGACAACAG CC - |
| #AGTACATG 1080 |
| - - AACTGGCTCG ACAGCGGCAA CGCCGGCCCC TGCAGCAGCA CCGAGGGCAA CC - |
| #CATCCAAC 1140 |
| - - ATCCTGGCCA ACAACCCCAA CACGCACGTC GTCTTCTCCA ACATCCGCTG GG - |
| #GAGACATT 1200 |
| - - GGGTCTACTA CGAACTCGAC TGCGCCCCCG CCCCCGCCTG CGTCCAGCAC GA - |
| #CGTTTTCG 1260 |
| - - ACTACACGGA GGAGCTCGAC GACTTCGAGC AGCCCGAGCT GCACGCAGAC TC - |
| #ACTGGGGG 1320 |
| - - CAGTGCGGTG GCATTGGGTA CAGCGGGTGC AAGACGTGCA CGTCGGGCAC TA - |
| #CGTGCCAG 1380 |
| - - TATAGCAACG ACTACTACTC GCAATGCCTT TAGAGCGTTG ACTTGCCTCT GG - |
| #TCTGTCCA 1440 |
| - - GACGGGGGCA CGATAGAATG CGGGCACGCA GGGAGCTCGT AGACATTGGG CT - |
| #TAATATAT 1500 |
| - - AAGACATGCT ATGTTGTATC TACATTAGCA AATGACAAAC AAATGAAAAA GA - |
| #ACTTATCA 1560 |
| - - AGCAAAAAAA AAAAAAAAAA AAAAAAAA - # - # |
| 1588 |
| - - - - (2) INFORMATION FOR SEQ ID NO:17: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 1820 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: |
| - - CCGCGGACTG CGCATCATGT ATCGGAAGTT GGCCGTCATC TCGGCCTTCT TG - |
| #GCCACAGC 60 |
| - - TCGTGCTCAG TCGGCCTGCA CTCTCCAATC GGAGACTCAC CCGCCTCTGA CA - |
| #TGGCAGAA 120 |
| - - ATGCTCGTCT GGTGGCACTT GCACTCAACA GACAGGCTCC GTGGTCATCG AC - |
| #GCCAACTG 180 |
| - - GCGCTGGACT CACGCTACGA ACAGCAGCAC GAACTGCTAC GATGGCAACA CT - |
| #TGGAGCTC 240 |
| - - GACCCTATGT CCTGACAACG AGACCTGCGC GAAGAACTGC TGTCTGGACG GT - |
| #GCCGCCTA 300 |
| - - CGCGTCCACG TACGGAGTTA CCACGAGCGG TAACAGCCTC TCCATTGGCT TT - |
| #GTCACCCA 360 |
| - - GTCTGCGCAG AAGAACGTTG GCGCTCGCCT TTACCTTATG GGCAGCGACA CG - |
| #ACCTACCA 420 |
| - - GGAATTCACC CTGCTTGGCA ACGAGTTCTC TTTCGATGTT GATGTTTCGC AG - |
| #CTGCCGTA 480 |
| - - AGTGACTTAC CATGAACCCC TGACGTATCT TCTTGTGGGC TCCCAGCTGA CT - |
| #GGCCAATT 540 |
| - - TAAGGTGCGG CTTGAACGGA GCTCTCTACT TCGTGTCCAT GGACGCGGAT GG - |
| #TGGCGTGA 600 |
| - - GCAAGTATCC CACCAACACC GCTGGCGCCA AGTACGGCAC GGGGTACTGT GA - |
| #CAGCCAGT 660 |
| - - GTCCCCGCGA TCTGAAGTTC ATCAATGGCC AGGCCAACGT TGAGGGCTGG GA - |
| #GCCGTCAT 720 |
| - - CCAACAACGC AAACACGGGC ATTGGAGGAC ACGGAAGCTG CTGCTCTGAG AT - |
| #GGATATCT 780 |
| - - GGGAGGCCAA CTCCATCTCC GAGGCTCTTA CCCCCCACCC TTGCACGACT GT - |
| #CGGCCAGG 840 |
| - - AGATCTGCGA GGGTGATGGG TGCGGCGGAA CTTACTCCGA TAACAGATAT GG - |
| #CGGCACTT 900 |
| - - GCGATCCCGA TGGCTGCGAC TGGAACCCAT ACCGCCTGGG CAACACCAGC TT - |
| #CTACGGCC 960 |
| - - CTGGCTCAAG CTTTACCCTC GATACCACCA AGAAATTGAC CGTTGTCACC CA - |
| #GTCCGAGA 1020 |
| - - CGTCGGGTGC CATCAACCGA TACTATGTCC AGAATGGCGT CACTTTCCAG CA - |
| #GCCCAACG 1080 |
| - - CCGAGCTTGG TAGTTACTCT GGCAACGAGC TCAACGATGA TTACTGCACA GC - |
| #TGAGGAGG 1140 |
| - - CAGAATTCGG CGGATCCTCT TTCTCAGACA AGGGCGGCCT GACTCAGTTC AA - |
| #GAAGGCTA 1200 |
| - - CCTCTGGCGG CATGGTTCTG GTCATGAGTC TGTGGGATGA TGTGAGTTTG AT - |
| #GGACAAAC 1260 |
| - - ATGCGCGTTG ACAAAGAGTC AAGCAGCTGA CTGAGATGTT ACAGTACTAC GC - |
| #CAACATGC 1320 |
| - - TGTGGCTGGA CTCCACCTAC CCGACAAACG AGACCTCCTC CACACCCGGT GC - |
| #CGTGCGCG 1380 |
| - - GAAGCTGCTC CACCAGCTCC GGTGTCCCTG CTCAGGTCGA ATCTCAGTCT CC - |
| #CAACGCCA 1440 |
| - - AGGTCACCTT CTCCAACATC AAGTTCGGAC CCATTGGCAG CACCGGCAAC CC - |
| #TAGCGGCG 1500 |
| - - GCAACCCTCC CGGCGGAAAC CCGCCTGGCA CCACCACCAC CCGCCGCCCA GC - |
| #CACTACCA 1560 |
| - - CTGGAAGCTC TCCCGGACCT ACCCAGTCTC ACTACGGCCA GTGCGGCGGT AT - |
| #TGGCTACA 1620 |
| - - GCGGCCCCAC GGTCTGCGCC AGCGGCACAA CTTGCCAGGT CCTGAACCCT TA - |
| #CTACTCTC 1680 |
| - - AGTGCCTGTA AAGCTCCGTG CGAAAGCCTG ACGCACCGGT AGATTCTTGG TG - |
| #AGCCCGTA 1740 |
| - - TCATGACGGC GGCGGGAGCT ACATGGCCCC GGGTGATTTA TTTTTTTTGT AT - |
| #CTACTTCT 1800 |
| - - GACCCTTTTC AAATATACGG - # - # |
| 182 - #0 |
| - - - - (2) INFORMATION FOR SEQ ID NO:18: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 2218 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: both |
| (D) TOPOLOGY: both |
| - - (ii) MOLECULE TYPE: cDNA |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: |
| - - GAATTCTCAC GGTGAATGTA GGCCTTTTGT AGGGTAGGAA TTGTCACTCA AG - |
| #CACCCCCA 60 |
| - - ACCTCCATTA CGCCTCCCCC ATAGAGTTCC CAATCAGTGA GTCATGGCAC TG - |
| #TTCTCAAA 120 |
| - - TAGATTGGGG AGAAGTTGAC TTCCGCCCAG AGCTGAAGGT CGCACAACCG CA - |
| #TGATATAG 180 |
| - - GGTCGGCAAC GGCAAAAAAG CACGTGGCTC ACCGAAAAGC AAGATGTTTG CG - |
| #ATCTAACA 240 |
| - - TCCAGGAACC TGGATACATC CATCATCACG CACGACCACT TTGATCTGCT GG - |
| #TAAACTCG 300 |
| - - TATTCGCCCT AAACCGAAGT GCGTGGTAAA TCTACACGTG GGCCCCTTTC GG - |
| #TATACTGC 360 |
| - - GTGTGTCTTC TCTAGGTGGC ATTCTTTTCC CTTCCTCTAG TGTTGAATTG TT - |
| #TGTGTTGG 420 |
| - - AGTCCGAGCT GTAACTACCT CTGAATCTCT GGAGAATGGT GGACTAACGA CT - |
| #ACCGTGCA 480 |
| - - CCTGCATCAT GTATATAATA GTGATCCTGA GAAGGGGGGT TTGGAGCAAT GT - |
| #GGGACTTT 540 |
| - - GATGGTCATC AAACAAAGAA CGAAGACGCC TCTTTTGCAA AGTTTTGTTT CG - |
| #GCTACGGT 600 |
| - - GAAGAACTGG ATACTTGTTG TGTCTTCTGT GTATTTTTGT GGCAACAAGA GG - |
| #CCAGAGAC 660 |
| - - AATCTATTCA AACACCAAGC TTGCTCTTTT GAGCTACAAG AACCTGTGGG GT - |
| #ATATATCT 720 |
| - - AGAGTTGTGA AGTCGGTAAT CCCGCTGTAT AGTAATACGA GTCGCATCTA AA - |
| #TACTCCGA 780 |
| - - AGCTGCTGCG AACCCGGAGA ATCGAGATGT GCTGGAAAGC TTCTAGCGAG CG - |
| #GCTAAATT 840 |
| - - AGCATGAAAG GCTATGAGAA ATTCTGGAGA CGGCTTGTTG AATCATGGCG TT - |
| #CCATTCTT 900 |
| - - CGACAAGCAA AGCGTTCCGT CGCAGTAGCA GGCACTCATT CCCGAAAAAA CT - |
| #CGGAGATT 960 |
| - - CCTAAGTAGC GATGGAACCG GAATAATATA ATAGGCAATA CATTGAGTTG CC - |
| #TCGACGGT 1020 |
| - - TGCAATGCAG GGGTACTGAG CTTGGACATA ACTGTTCCGT ACCCCACCTC TT - |
| #CTCAACCT 1080 |
| - - TTGGCGTTTC CCTGATTCAG CGTACCCGTA CAAGTCGTAA TCACTATTAA CC - |
| #CAGACTGA 1140 |
| - - CCGGACGTGT TTTGCCCTTC ATTTGGAGAA ATAATGTCAT TGCGATGTGT AA - |
| #TTTGCCTG 1200 |
| - - CTTGACCGAC TGGGGCTGTT CGAAGCCCGA ATGTAGGATT GTTATCCGAA CT - |
| #CTGCTCGT 1260 |
| - - AGAGGCATGT TGTGAATCTG TGTCGGGCAG GACACGCCTC GAAGGTTCAC GG - |
| #CAAGGGAA 1320 |
| - - ACCACCGATA GCAGTGTCTA GTAGCAACCT GTAAAGCCGC AATGCAGCAT CA - |
| #CTGGAAAA 1380 |
| - - TACAAACCAA TGGCTAAAAG TACATAAGTT AATGCCTAAA GAAGTCATAT AC - |
| #CAGCGGCT 1440 |
| - - AATAATTGTA CAATCAAGTG GCTAAACGTA CCGTAATTTG CCAACGGCTT GT - |
| #GGGGTTGC 1500 |
| - - AGAAGCAACG GCAAAGCCCC ACTTCCCCAC GTTTGTTTCT TCACTCAGTC CA - |
| #ATCTCAGC 1560 |
| - - TGGTGATCCC CCAATTGGGT CGCTTGTTTG TTCCGGTGAA GTGAAAGAAG AC - |
| #AGAGGTAA 1620 |
| - - GAATGTCTGA CTCGGAGCGT TTTGCATACA ACCAAGGGCA GTGATGGAAG AC - |
| #AGTGAAAT 1680 |
| - - GTTGACATTC AAGGAGTATT TAGCCAGGGA TGCTTGAGTG TATCGTGTAA GG - |
| #AGGTTTGT 1740 |
| - - CTGCCGATAC GACGAATACT GTATAGTCAC TTCTGATGAA GTGGTCCATA TT - |
| #GAAATGTA 1800 |
| - - AGTCGGCACT GAACAGGCAA AAGATTGAGT TGAAACTGCC TAAGATCTCG GG - |
| #CCCTCGGG 1860 |
| - - CCTTCGGCCT TTGGGTGTAC ATGTTTGTGC TCCGGGCAAA TGCAAAGTGT GG - |
| #TAGGATCG 1920 |
| - - AACACACTGC TGCCTTTACC AAGCAGCTGA GGGTATGTGA TAGGCAAATG TT - |
| #CAGGGGCC 1980 |
| - - ACTGCATGGT TTCGAATAGA AAGAGAAGCT TAGCCAAGAA CAATAGCCGA TA - |
| #AAGATAGC 2040 |
| - - CTCATTAAAC GGAATGAGCT AGTAGGCAAA GTCAGCGAAT GTGTATATAT AA - |
| #AGGTTCGA 2100 |
| - - GGTCCGTGCC TCCCTCATGC TCTCCCCATC TACTCATCAA CTCAGATCCT CC - |
| #AGGAGACT 2160 |
| - - TGTACACCAT CTTTTGAGGC ACAGAAACCC AATAGTCAAC CGCGGACTGC GC - |
| #ATCATG 2218 |
| - - - - (2) INFORMATION FOR SEQ ID NO:19: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 1142 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: both |
| (D) TOPOLOGY: both |
| - - (ii) MOLECULE TYPE: cDNA |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: |
| - - GAATTCTCAC GGTGAATGTA GGCCTTTTGT AGGGTAGGAA TTGTCACTCA AG - |
| #CACCCCCA 60 |
| - - ACCTCCATTA CGCCTCCCCC ATAGAGTTCC CAATCAGTGA GTCATGGCAC TG - |
| #TTCTCAAA 120 |
| - - TAGATTGGGG AGAAGTTGAC TTCCGCCCAG AGCTGAAGGT CGCACAACCG CA - |
| #TGATATAG 180 |
| - - GGTCGGCAAC GGCAAAAAAG CACGTGGCTC ACCGAAAAGC AAGATGTTTG CG - |
| #ATCTAACA 240 |
| - - TCCAGGAACC TGGATACATC CATCATCACG CACGACCACT TTGATCTGCT GG - |
| #TAAACTCG 300 |
| - - TATTCGCCCT AAACCGAAGT GCGTGGTAAA TCTACACGTG GGCCCCTTTC GG - |
| #TATACTGC 360 |
| - - GTGTGTCTTC TCTAGGTGGC ATTCTTTTCC CTTCCTCTAG TGTTGAATTG TT - |
| #TGTGTTGG 420 |
| - - AGTCCGAGCT GTAACTACCT CTGAATCTCT GGAGAATGGT GGACTAACGA CT - |
| #ACCGTGCA 480 |
| - - CCTGCATCAT GTATATAATA GTGATCCTGA GAAGGGGGGT TTGGAGCAAT GT - |
| #GGGACTTT 540 |
| - - GATGGTCATC AAACAAAGAA CGAAGACGCC TCTTTTGCAA AGTTTTGTTT CG - |
| #GCTACGGT 600 |
| - - GAAGAACTGG ATACTTGTTG TGTCTTCTGT GTATTTTTGT GGCAACAAGA GG - |
| #CCAGAGAC 660 |
| - - AATCTATTCA AACACCAAGC TTGCTCTTTT GAGCTACAAG AACCTGTGGG GT - |
| #ATATATCT 720 |
| - - AGTGGCCAGA ATGCCTAGGT CACCTCTAGA GAGTTGAAAC TGCCTAAGAT CT - |
| #CGGGCCCT 780 |
| - - CGGGCCTTCG GCCTTTGGGT GTACATGTTT GTGCTCCGGG CAAATGCAAA GT - |
| #GTGGTAGG 840 |
| - - ATCGAACACA CTGCTGCCTT TACCAAGCAG CTGAGGGTAT GTGATAGGCA AA - |
| #TGTTCAGG 900 |
| - - GGCCACTGCA TGGTTTCGAA TAGAAAGAGA AGCTTAGCCA AGAACAATAG CC - |
| #GATAAAGA 960 |
| - - TAGCCTCATT AAACGGAATG AGCTAGTAGG CAAAGTCAGC GAATGTGTAT AT - |
| #ATAAAGGT 1020 |
| - - TCGAGGTCCG TGCCTCCCTC ATGCTCTCCC CATCTACTCA TCAACTCAGA TC - |
| #CTCCAGGA 1080 |
| - - GACTTGTACA CCATCTTTTG AGGCACAGAA ACCCAATAGT CAACCGCGGA CT - |
| #GCGCATCA 1140 |
| - - TG - # - # - # |
| 1142 |
| - - - - (2) INFORMATION FOR SEQ ID NO:20: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 2266 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: both |
| (D) TOPOLOGY: both |
| - - (ii) MOLECULE TYPE: cDNA |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: |
| - - GAATTCTCAC GGTGAATGTA GGCCTTTTGT AGGGTAGGAA TTGTCACTCA AG - |
| #CACCCCCA 60 |
| - - ACCTCCATTA CGCCTCCCCC ATAGAGTTCC CAATCAGTGA GTCATGGCAC TG - |
| #TTCTCAAA 120 |
| - - TAGATTGGGG AGAAGTTGAC TTCCGCCCAG AGCTGAAGGT CGCACAACCG CA - |
| #TGATATAG 180 |
| - - GGTCGGCAAC GGCAAAAAAG CACGTGGCTC ACCGAAAAGC AAGATGTTTG CG - |
| #ATCTAACA 240 |
| - - TCCAGGAACC TGGATACATC CATCATCACG CACGACCACT TTGATCTGCT GG - |
| #TAAACTCG 300 |
| - - TATTCGCCCT AAACCGAAGT GCGTGGTAAA TCTACACGTG GGCCCCTTTC GG - |
| #TATACTGC 360 |
| - - GTGTGTCTTC TCTAGGTGGC ATTCTTTTCC CTTCCTCTAG TGTTGAATTG TT - |
| #TGTGTTGG 420 |
| - - AGTCCGAGCT GTAACTACCT CTGAATCTCT GGAGAATGGT GGACTAACGA CT - |
| #ACCGTGCA 480 |
| - - CCTGCATCAT GTATATAATA GTGATCCTGA GAAGGGGGGT TTGGAGCAAT GT - |
| #GGGACTTT 540 |
| - - GATGGTCATC AAACAAAGAA CGAAGACGCC TCTTTTGCAA AGTTTTGTTT CG - |
| #GCTACGGT 600 |
| - - GAAGAACTGG ATACTTGTTG TGTCTTCTGT GTATTTTTGT GGCAACAAGA GG - |
| #CCAGAGAC 660 |
| - - AATCTATTCA AACACCAAGC TTGCTCTTTT GAGCTACAAG AACCTGTGGG GT - |
| #ATATATCT 720 |
| - - AGTGGCCAGA ATGCCTAGGT CACCTCTAAA GGTACCCTGC AGCTCGAGCT AG - |
| #AGTTGTGA 780 |
| - - AGTCGGTAAT CCCGCTGTAT AGTAATACGA GTCGCATCTA AATACTCCGA AG - |
| #CTGCTGCG 840 |
| - - AACCCGGAGA ATCGAGATGT GCTGGAAAGC TTCTAGCGAG CGGCTAAATT AG - |
| #CATGAAAG 900 |
| - - GCTATGAGAA ATTCTGGAGA CGGCTTGTTG AATCATGGCG TTCCATTCTT CG - |
| #ACAAGCAA 960 |
| - - AGCGTTCCGT CGCAGTAGCA GGCACTCATT CCCGAAAAAA CTCGGAGATT CC - |
| #TAAGTAGC 1020 |
| - - GATGGAACCG GAATAATATA ATAGGCAATA CATTGAGTTG CCTCGACGGT TG - |
| #CAATGCAG 1080 |
| - - GGGTACTGAG CTTGGACATA ACTGTTCCGT ACCCCACCTC TTCTCAACCT TT - |
| #GGCGTTTC 1140 |
| - - CCTGATTCAG CGTACCCGTA CAAGTCGTAA TCACTATTAA CCCAGACTGA CC - |
| #GGACGTGT 1200 |
| - - TTTGCCCTTC ATTTGGAGAA ATAATGTCAT TGCGATGTGT AATTTGCCTG CT - |
| #TGACCGAC 1260 |
| - - TGGGGCTGTT CGAAGCCCGA ATGTAGGATT GTTATCCGAA CTCTGCTCGT AG - |
| #AGGCATGT 1320 |
| - - TGTGAATCTG TGTCGGGCAG GACACGCCTC GAAGGTTCAC GGCAAGGGAA AC - |
| #CACCGATA 1380 |
| - - GCAGTGTCTA GTAGCAACCT GTAAAGCCGC AATGCAGCAT CACTGGAAAA TA - |
| #CAAACCAA 1440 |
| - - TGGCTAAAAG TACATAAGTT AATGCCTAAA GAAGTCATAT ACCAGCGGCT AA - |
| #TAATTGTA 1500 |
| - - CAATCAAGTG GCTAAACGTA CCGTAATTTG CCAACGCGTT TCTAGATTGC AG - |
| #AAGCACGG 1560 |
| - - CAAAGCCCAC TTACCCACGT TTGTTTCTTC ACTCAGTCCA ATCTCAGCTG GT - |
| #GATCCCCC 1620 |
| - - AATTGGGTCG CTTGTTTGTT CCGGTGAAGT GAAAGAAGAC AGAGGTAAGA AT - |
| #GTCTGACT 1680 |
| - - CGGAGCGTTT TGCATACAAC CAAGGGCAGT GATGGAAGAC AGTGAAATGT TG - |
| #ACATTCAA 1740 |
| - - GGAGTATTTA GCCAGGGATG CTTGAGTGTA TCGTGTAAGG AGGTTTGTCT GC - |
| #CGATACGA 1800 |
| - - CGAATACTGT ATAGTCACTT CTGATGAAGT GGTCCATATT GAAATGTAAG TC - |
| #GGCACTGA 1860 |
| - - ACAGGCAAAA GATTGAGTTG AAACTGCCTA AGATCTCGGG CCCTCGGGCC TT - |
| #CGGCCTTT 1920 |
| - - GGGTGTACAT GTTTGTGCTC CGGGCAAATG CAAAGTGTGG TAGGATCGAA CA - |
| #CACTGCTG 1980 |
| - - CCTTTACCAA GCAGCTGAGG GTATGTGATA GGCAAATGTT CAGGGGCCAC TG - |
| #CATGGTTT 2040 |
| - - CGAATAGAAA GAGAAGCTTA GCCAAGAACA ATAGCCGATA AAGATAGCCT CA - |
| #TTAAACGG 2100 |
| - - AATGAGCTAG TAGGCAAAGT CAGCGAATGT GTATATATAA AGGTTCGAGG TC - |
| #CGTGCCTC 2160 |
| - - CCTCATGCTC TCCCCATCTA CTCATCAACT CAGATCCTCC AGGAGACTTG TA - |
| #CACCATCT 2220 |
| - - TTTGAGGCAC AGAAACCCAA TAGTCAACCG CGGACTGCGC ATCATG - # |
| 2266 |
| - - - - (2) INFORMATION FOR SEQ ID NO:21: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 1781 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: both |
| (D) TOPOLOGY: both |
| - - (ii) MOLECULE TYPE: cDNA |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: |
| - - CAATTCTCAC GGTGAATGTA GGCCTTTTGT AGGGTAGGAA TTGTCACTCA AG - |
| #CACCCCCA 60 |
| - - ACCTCCATTA CGCCTCCCCC ATAGAGTTCC CAATCAGTGA GTCATGGCAC TG - |
| #TTCTCAAA 120 |
| - - TAGATTGGGG AGAAGTTGAC TTCCGCCCAG AGCTGAAGGT CGCACAACCG CA - |
| #TGATATAG 180 |
| - - GGTCGGCAAC GGCAAAAAAG CACGTGGCTC ACCGAAAAGC AAGATGTTTG CG - |
| #ATCTAACA 240 |
| - - TCCAGGAACC TGGATACATC CATCATCACG CACGACCACT TTGATCTGCT GG - |
| #TAAACTCG 300 |
| - - TATTCGCCCT AAACCGAAGT GCGTGGTAAA TCTACACGTG GGCCCCTTTC GG - |
| #TATACTGC 360 |
| - - GTGTGTCTTC TCTAGGTGGC ATTCTTTTCC CTTCCTCTAG TGTTGAATTG TT - |
| #TGTGTTGG 420 |
| - - AGTCCGAGCT GTAACTACCT CTGAATCTCT GGAGAATGGT GGACTAACGA CT - |
| #ACCGTGCA 480 |
| - - CCTGCATCAT GTATATAATA GTGATCCTGA GAAGGGGGGT TTGGAGCAAT GT - |
| #GGGACTTT 540 |
| - - GATGGTCATC AAACAAAGAA CGAAGACGCC TCTTTTGCAA AGTTTTGTTT CG - |
| #GCTACGGT 600 |
| - - GAAGAACTGG ATACTTGTTG TGTCTTCTGT GTATTTTTGT GGCAACAAGA GG - |
| #CCAGAGAC 660 |
| - - AATCTATTCA AACACCAAGC TTGCTCTTTT GAGCTACAAG AACCTTCTAA AT - |
| #ATATATCT 720 |
| - - AGTGGCCAGA ATGCCTAGGT CACCTCTAAA TGTGTAATTT GCCTGCTTGA CC - |
| #GACTGGGG 780 |
| - - CTGTTCGAAG CCCGAATGTA GGATTGTTAT CCGAACTCTG CTCGTAGAGG CA - |
| #TGTTGTGA 840 |
| - - ATCTGTGTCG GGCAGGACAC GCCTCGAAGG TTCACGGCAA GGGAAACCAC CG - |
| #ATAGCAGT 900 |
| - - GTCTAGTAGC AACCTGTAAA GCCGCAATGC AGCATCACTG GAAAATACAA AC - |
| #CAATGGCT 960 |
| - - AAAAGTACAT AAGTTAATGC CTAAAGAAGT CATATACCAG CGGCTAATAA TT - |
| #GTACAATC 1020 |
| - - AAGTGGCTAA ACGTACCGTA ATTTGCCAAC GCGTTTCTAG ATTGCAGAAG CA - |
| #CGGCAAAG 1080 |
| - - CCCACTTACC CACGTTTGTT TCTTCACTCA GTCCAATCTC AGCTGGTGAT CC - |
| #CCCAATTG 1140 |
| - - GGTCGCTTGT TTGTTCCGGT GAAGTGAAAG AAGACAGAGG TAAGAATGTC TG - |
| #ACTCGGAG 1200 |
| - - CGTTTTGCAT ACAACCAAGG GCAGTGATGG AAGACAGTGA AATGTTGACA TT - |
| #CAAGGAGT 1260 |
| - - ATTTAGCCAG GGATGCTTGA GTGTATCGTG TAAGGAGGTT TGTCTGCCGA TA - |
| #CGACGAAT 1320 |
| - - ACTGTATAGT CACTTCTGAT GAAGTGGTCC ATATTGAAAT GTAAGTCGGC AC - |
| #TGAACAGG 1380 |
| - - CAAAAGATTG AGTTGAAACT GCCTAAGATC TCGGGCCCTC GGGCCTTCGG CC - |
| #TTTGGGTG 1440 |
| - - TACATGTTTG TGCTCCGGGC AAATGCAAAG TGTGGTAGGA TCGAACACAC TG - |
| #CTGCCTTT 1500 |
| - - ACCAAGCAGC TGAGGGTATG TGATAGGCAA ATGTTCAGGG GCCACTGCAT GG - |
| #TTTCGAAT 1560 |
| - - AGAAAGAGAA GCTTAGCCAA GAACAATAGC CGATAAAGAT AGCCTCATTA AA - |
| #CGGAATGA 1620 |
| - - GCTAGTAGGC AAAGTCAGCG AATGTGTATA TATAAAGGTT CGAGGTCCGT GC - |
| #CTCCCTCA 1680 |
| - - TGCTCTCCCC ATCTACTCAT CAACTCAGAT CCTCCAGGAG ACTTGTACAC CA - |
| #TCTTTTGA 1740 |
| - - GGCACAGAAA CCCAATAGTC AACCGCGGAC TGCGCATCAT G - # |
| - # 1781 |
| - - - - (2) INFORMATION FOR SEQ ID NO:22: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 1781 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: both |
| (D) TOPOLOGY: both |
| - - (ii) MOLECULE TYPE: cDNA |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: |
| - - CAATTCTCAC GGTGAATGTA GGCCTTTTGT AGGGTAGGAA TTGTCACTCA AG - |
| #CACCCCCA 60 |
| - - ACCTCCATTA CGCCTCCCCC ATAGAGTTCC CAATCAGTGA GTCATGGCAC TG - |
| #TTCTCAAA 120 |
| - - TAGATTGGGG AGAAGTTGAC TTCCGCCCAG AGCTGAAGGT CGCACAACCG CA - |
| #TGATATAG 180 |
| - - GGTCGGCAAC GGCAAAAAAG CACGTGGCTC ACCGAAAAGC AAGATGTTTG CG - |
| #ATCTAACA 240 |
| - - TCCAGGAACC TGGATACATC CATCATCACG CACGACCACT TTGATCTGCT GG - |
| #TAAACTCG 300 |
| - - TATTCGCCCT AAACCGAAGT GCGTGGTAAA TCTACACGTG GGCCCCTTTC GG - |
| #TATACTGC 360 |
| - - GTGTGTCTTC TCTAGGTGGC ATTCTTTTCC CTTCCTCTAG TGTTGAATTG TT - |
| #TGTGTTGG 420 |
| - - AGTCCGAGCT GTAACTACCT CTGAATCTCT GGAGAATGGT GGACTAACGA CT - |
| #ACCGTGCA 480 |
| - - CCTGCATCAT GTATATAATA GTGATCCTGA GAAGGGGGGT TTGGAGCAAT GT - |
| #GGGACTTT 540 |
| - - GATGGTCATC AAACAAAGAA CGAAGACGCC TCTTTTGCAA AGTTTTGTTT CG - |
| #GCTACGGT 600 |
| - - GAAGAACTGG ATACTTGTTG TGTCTTCTGT GTATTTTTGT GGCAACAAGA GG - |
| #CCAGAGAC 660 |
| - - AATCTATTCA AACACCAAGC TTGCTCTTTT GAGCTACAAG AACCTTCTAA AT - |
| #ATATATCT 720 |
| - - AGTGGCCAGA ATGCCTAGGT CACCTCTAAA TGTGTAATTT GCCTGCTTGA CC - |
| #GATCTAAA 780 |
| - - CTGTTCGAAG CCCGAATGTA GGATTGTTAT CCGAACTCTG CTCGTAGAGG CA - |
| #TGTTGTGA 840 |
| - - ATCTGTGTCG GGCAGGACAC GCCTCGAAGG TTCACGGCAA GGGAAACCAC CG - |
| #ATAGCAGT 900 |
| - - GTCTAGTAGC AACCTGTAAA GCCGCAATGC AGCATCACTG GAAAATACAA AC - |
| #CAATGGCT 960 |
| - - AAAAGTACAT AAGTTAATGC CTAAAGAAGT CATATACCAG CGGCTAATAA TT - |
| #GTACAATC 1020 |
| - - AAGTGGCTAA ACGTACCGTA ATTTGCCAAC GCGTTTCTAG ATTGCAGAAG CA - |
| #CGGCAAAG 1080 |
| - - CCCACTTACC CACGTTTGTT TCTTCACTCA GTCCAATCTC AGCTGGTGAT CC - |
| #CCCAATTG 1140 |
| - - GGTCGCTTGT TTGTTCCGGT GAAGTGAAAG AAGACAGAGG TAAGAATGTC TG - |
| #ACTCGGAG 1200 |
| - - CGTTTTGCAT ACAACCAAGG GCAGTGATGG AAGACAGTGA AATGTTGACA TT - |
| #CAAGGAGT 1260 |
| - - ATTTAGCCAG GGATGCTTGA GTGTATCGTG TAAGGAGGTT TGTCTGCCGA TA - |
| #CGACGAAT 1320 |
| - - ACTGTATAGT CACTTCTGAT GAAGTGGTCC ATATTGAAAT GTAAGTCGGC AC - |
| #TGAACAGG 1380 |
| - - CAAAAGATTG AGTTGAAACT GCCTAAGATC TCGGGCCCTC GGGCCTTCGG CC - |
| #TTTGGGTG 1440 |
| - - TACATGTTTG TGCTCCGGGC AAATGCAAAG TGTGGTAGGA TCGAACACAC TG - |
| #CTGCCTTT 1500 |
| - - ACCAAGCAGC TGAGGGTATG TGATAGGCAA ATGTTCAGGG GCCACTGCAT GG - |
| #TTTCGAAT 1560 |
| - - AGAAAGAGAA GCTTAGCCAA GAACAATAGC CGATAAAGAT AGCCTCATTA AA - |
| #CGGAATGA 1620 |
| - - GCTAGTAGGC AAAGTCAGCG AATGTGTATA TATAAAGGTT CGAGGTCCGT GC - |
| #CTCCCTCA 1680 |
| - - TGCTCTCCCC ATCTACTCAT CAACTCAGAT CCTCCAGGAG ACTTGTACAC CA - |
| #TCTTTTGA 1740 |
| - - GGCACAGAAA CCCAATAGTC AACCGCGGAC TGCGCATCAT G - # |
| - # 1781 |
| - - - - (2) INFORMATION FOR SEQ ID NO:23: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 745 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: |
| - - GGACCTACCC AGTCTCACTA CGGCCAGTGC GGCGGTATTG GCTACAGCGG CC - |
| #CCACGGTC 60 |
| - - TGCGCCAGCG GCACAACTTG CCAGGTCCTG AACCCTTACT ACTCTCAGTG CC - |
| #TGTAAAGC 120 |
| - - TCCGTGCGAA AGCCTGACGC ACCGGTAGAT TCTTGGTGAG CCCGTATCAT GA - |
| #CGGCGGCG 180 |
| - - GGAGCTACAT GGCCCCGGGT GATTTATTTT TTTTGTATCT ACTTCTGACC CT - |
| #TTTCAAAT 240 |
| - - ATACGGTCAA CTCATCTTTC ACTGGAGATG CGGCCTGCTT GGTATTGCGA TG - |
| #TTGTCAGC 300 |
| - - TTGGCAAATT GTGGCTTTCG AAAACACAAA ACGATTCCTT AGTAGCCATG CA - |
| #TTTTAAGA 360 |
| - - TAACGGAATA GAAGAAAGAG GAAATTAAAA AAAAAAAAAA AACAAACATC CC - |
| #GTTCATAA 420 |
| - - CCCGTAGAAT CGCCGCTCTT CGTGTATCCC AGTACCACGT CAAAGGTATT CA - |
| #TGATCGTT 480 |
| - - CAATGTTGAT ATTGTTCCGC CAGTATGGCT CCACCCCCAT CTCCGCGAAT CT - |
| #CCTCTTCT 540 |
| - - CGAACGCGGT AGTGGCTGCT GCCAATTGGT AATGACCATA GGGAGACAAA CA - |
| #GCATAATA 600 |
| - - GCAACAGTGG AAATTAGTGG CGCAATAATT GAGAACACAG TGAGACCATA GC - |
| #TGGCGGCC 660 |
| - - TGGAAAGCAC TGTTGGAGAC CAACTTGTCC GTTGCGAGGC CAACTTGCAT TG - |
| #CTGTCAAG 720 |
| - - ACGATGACAA CGTAGCCGAG GACCC - # - # |
| 745 |
| - - - - (2) INFORMATION FOR SEQ ID NO:24: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 1627 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: |
| - - GGCGGTATTG GCTACAGCGG CCCCACGGTC TGCGCCAGCG GCACAACTTG CC - |
| #AGGTCCTG 60 |
| - - AACCCTTACT ACTCTCAGTG CCTGTAAAGC TCCGTGCGAA AGCCTGACGC AC - |
| #CGGTAGAT 120 |
| - - TCTTGGTGAG CCCGTATCAT GACGGCGGCG GGAGCTACAT GGCCCCGGGT GA - |
| #TTTATTTT 180 |
| - - TTTTGTATCT ACTTCTGACC CTTTTCAAAT ATACGGTCAA CTCATCTTTC AC - |
| #TGGAGATG 240 |
| - - CGGCCTGCTT GGTATTGCGA TGTTGTCAGC TTGGCAAATT GTGGCTTTCG AA - |
| #AACACAAA 300 |
| - - ACGATTCCTT AGTAGCCATG CATCGGGATC CTTTAAGATA ACGGAATAGA AG - |
| #AAAGAGGA 360 |
| - - AATTAAAAAA AAAAAAAAAA CAAACATCCC GTTCATAACC CGTAGAATCG CC - |
| #GCTCTTCG 420 |
| - - TGTATCCCAG TACCACGGCA AAGGTATTTC ATGATCGTTC AATGTTGATA TT - |
| #GTTCCCGC 480 |
| - - CAGTATGGCT GCACCCCCAT CTCCGCGAAT CTCCTCTTCT CGAACGCGGT AG - |
| #TGGCGCGC 540 |
| - - CAATTGGTAA TGACCATAGG GAGACAAACA GCATAATAGC AACAGTGGAA AT - |
| #TAGTGGCG 600 |
| - - CAATAATTGA GAACACAGTG AGACCATAGC TGGCGGCCTG GAAAGCACTG TT - |
| #GGAGACCA 660 |
| - - ACTTGTCCGT TGCGAGGCCA ACTTGCATTG CTGTCAAGAC GATGACAACG TA - |
| #GCCGAGGA 720 |
| - - CCGTCACAAG GGACGCAAAG TTGTCGCGGA TGAGGTCTCC GTAGATGGCA TA - |
| #GCCGGCAA 780 |
| - - TCCGAGAGTA GCCTCTCAAC AGGTGGCCTT TTCGAAACCG GTAAACCTTG TT - |
| #CAGACGTC 840 |
| - - CTAGCCGCAG CTCACCGTAC CAGTATCGAG GATTGACGGC AGAATAGCAG TG - |
| #GCTCTCCA 900 |
| - - GGATTTGACT GGACAAAATC TTCCAGTATT CCCAGGTCAC AGTGTCTGGC AG - |
| #AAGTCCCT 960 |
| - - TCTCGCGTGC ANTCGAAAGT CGCTATAGTG CGCAATGAGA GCACAGTAGG AG - |
| #AATAGGAA 1020 |
| - - CCCGCGAGCA CATTGTTCAA TCTCCACATG AATTGGATGA CTGCTGGGCA GA - |
| #ATGTGCTG 1080 |
| - - CCTCCAAAAT CCTGCGTCCA ACAGATACTC TGGCAGGGGC TTCAGATGAA TG - |
| #CCTCTGGG 1140 |
| - - CCCCCAGATA AGATGCAGCT CTGGATTCTC GGTTACNATG ATATCGCGAG AG - |
| #AGCACGAG 1200 |
| - - TTGGTGATGG AGGGACAGGA GGCATAGGTC GCGCAGGCCC ATAACCAGTC TT - |
| #GCACAGCA 1260 |
| - - TTGATCTTAC CTCACGAGGA GCTCCTGATG CAGAAACTCC TCCATGTTGC TG - |
| #ATTGGGTT 1320 |
| - - GAGAATTTCA TCGCTCCTGG ATCGTATGGT TGCTGGCAAG ACCCTGCTTA AC - |
| #CGTGCCGT 1380 |
| - - GTCATGGTCA TCTCTGGTGG CTTCGTCGCT GGCCTGTCTT TGCAATTCGA CA - |
| #GCAAATGG 1440 |
| - - TGGAGATCTC TCTATCGTGA CAGTCATGGT AGCGATAGCT AGGTGTCGTT GC - |
| #ACGCACAT 1500 |
| - - AGGCCGAAAT GCGAAGTGGA AAGAATTTCC CGGNTGCGGA ATGAAGTCTC GT - |
| #CATTTTGT 1560 |
| - - ACTCGTACTC GACACCTCCA CCGAAGTGTT AATAATGGAT CCACGATGCC AA - |
| #AAAGCTTG 1620 |
| - - TGCATGC - # - # |
| - # 1627 |
| - - - - (2) INFORMATION FOR SEQ ID NO:25: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 91 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: |
| - - GGACTGGCAT CATGGCGCCC TCAGTTACAC TGCCGTTGAC CACGGCCATC CT - |
| #GGCCATTG 60 |
| - - CCCGGCTCGT CGCCGCCCAG CAACCGGGTA C - # - # |
| 91 |
| - - - - (2) INFORMATION FOR SEQ ID NO:26: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 97 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| - - (ix) FEATURE: |
| (A) NAME/KEY: CDS |
| (B) LOCATION: 18..95 |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: |
| - - AACCGCGGAC TGGCATC ATG GCG CCC TCA GTT ACA CTG - #CCG TTG ACC ACG |
| 50 |
| - # Met Ala Pro Ser Val Thr Leu Pro Leu Thr - #Thr |
| - # 1 - #5 - #10 |
| - - GCC ATC CTG GCC ATT GCC CGG CTC GTC GCC GC - #C CAG CAA CCG GGT |
| - #95 |
| Ala Ile Leu Ala Ile Ala Arg Leu Val Ala Al - #a Gln Gln Pro Gly |
| 15 - # 20 - # 25 |
| - - AC - # - # - # |
| 97 |
| - - - - (2) INFORMATION FOR SEQ ID NO:27: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 26 amino - #acids |
| (B) TYPE: amino acid |
| (D) TOPOLOGY: linear |
| - - (ii) MOLECULE TYPE: protein |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: |
| - - Met Ala Pro Ser Val Thr Leu Pro Leu Thr Th - #r Ala Ile Leu Ala Ile |
| 1 5 - # 10 - # 15 |
| - - Ala Arg Leu Val Ala Ala Gln Gln Pro Gly |
| 20 - # 25 |
| - - - - (2) INFORMATION FOR SEQ ID NO:28: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 15 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: |
| - - ACT ACG TAG TCG ACT - # - # |
| - # 15 |
| - - - - (2) INFORMATION FOR SEQ ID NO:29: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 50 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: double |
| (D) TOPOLOGY: linear |
| - - (ii) MOLECULE TYPE: cDNA |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: |
| - - CTAGTGGCCA GAATGCCTAG GTCACCTCTA GAGGTACCCT GCAGCTCGAG - # |
| 50 |
| - - - - (2) INFORMATION FOR SEQ ID NO:30: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 50 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: double |
| (D) TOPOLOGY: linear |
| - - (ii) MOLECULE TYPE: cDNA |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: |
| - - CTAGCTCGAG CTGCAGGGTA CCTCTAGAGG TGACCTAGGC ATTCTGGCCA - # |
| 50 |
| - - - - (2) INFORMATION FOR SEQ ID NO:31: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 18 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| - - (ii) MOLECULE TYPE: cDNA |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: |
| - - GGACTGCGCA TCATGCAG - # - # |
| - # 18 |
| - - - - (2) INFORMATION FOR SEQ ID NO:32: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 24 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: double |
| (D) TOPOLOGY: linear |
| - - (ii) MOLECULE TYPE: cDNA |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: |
| - - GATCCTGCAT GATGCGCAGT CCGC - # - # |
| 24 |
| - - - - (2) INFORMATION FOR SEQ ID NO:33: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 12 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: double |
| (D) TOPOLOGY: linear |
| - - (ii) MOLECULE TYPE: cDNA |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: |
| - - AATTCGTCGA CG - # - # |
| - # 12 |
| - - - - (2) INFORMATION FOR SEQ ID NO:34: |
| - - (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 22 base - #pairs |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: double |
| (D) TOPOLOGY: linear |
| - - (ii) MOLECULE TYPE: cDNA |
| - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: |
| - - TAATAATAAC CGGCGGTATT GG - # - # |
| 22 |
| __________________________________________________________________________ |
Nevalainen, Kaisu Milja Helena, Nakari, Tiina Hannele, Onnela, Maija-Leena, Ilmen, Marja Hannele, Penttila , Merja Elisa
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